Anti-garp protein and uses thereof

ABSTRACT

The present invention relates to a protein binding to GARP in the presence of TGF-β and uses thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/013,706, filed Feb. 2, 2016, which claims priority from U.S.Provisional Application No. 62/111,429, filed on Feb. 3, 2015, thecontents of each of which are hereby incorporated herein by reference intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety.

Said ASCII copy, created on Mar. 29, 2019, is named AbbVie_465C1_SL.txtand is 54,402 bytes in size.

FIELD OF INVENTION

The present invention relates to human anti-GARP protein that inhibitsTGF-β signaling. The present invention also relates to the treatment ofimmune disorders and diseases such as cancer.

BACKGROUND OF INVENTION

Since the molecular identification of the first human tumor antigens inthe early 1990's, several clinical trials were completed to evaluate theeffects of therapeutic vaccination of cancer patients with sharedtumor-specific antigens (Boon, T. et al. Annu. Rev. Immunol. 2006,24:175-208). Evidence of tumor regression was observed in about 20% ofthe patients, with objective clinical responses in 5-10%. Therefore,vaccination with tumor-specific antigens represents a new promisingtherapy for treating cancer.

Strategies are needed to improve the proportion of patients that respondto vaccination. The main limiting factor to clinical efficacy of currenttherapeutic cancer vaccines does not appear to be the vaccine itself,but local factors controlling the tumor microenvironment in which theanti-tumor T cells have to work.

Regulatory T cells, or Tregs, are a subset of CD4+ T lymphocytesspecialized in the inhibition of immune responses. Insufficient Tregfunction results in autoimmune pathology, while excessive Treg functionmay inhibit anti-tumor immune responses in cancer patients. The exactmechanisms by which Tregs inhibit immune responses are not fullyunderstood.

Due to their immunosuppressive functions, Tregs represent potentialinhibitors of spontaneous or vaccine-induced anti-tumor immuneresponses. In murine models, the depletion of Tregs can improve immuneresponses against experimental tumors (Colombo et al. Nat. Rev. Cancer2007, 7:880-887). Thus, targeting Tregs in humans could improve theefficacy of immunotherapy against cancer.

It has been demonstrated that active TGF-β is produced by human Tregs,but not other types of human T lymphocytes (Stockis, J. et al. Eur. J.Immunol. 2009, 39:869-882), TGF-β could be a target of interest.

However, antibodies against hTGF-β were not found promising. Phase 1clinical trials have been conducted in focal segmentalglomerulosclerosis (FSGS), idiopathic pulmonary fibrosis (IPF) andadvanced malignant melanoma or renal cell carcinoma (RCC) (Lonning S etal. Current Pharmaceutical Biotechnology 2011, 12:2176-2189). Dependingon the trial, adverse events were observed in some patients. The mainadverse reactions reported consisted in the development ofkeratoacanthoma (KA) and squamous cell carcinoma (SCC) in melanomapatients. It is possible that the KA or SCC lesions in melanoma patientsevolved from pre-cancerous cells whose proliferation was being inhibitedby endogenous TGF-β (Lonning S et al. Current PharmaceuticalBiotechnology 2011, 12:2176-2189). Therefore, a major concern regardingthe use of anti-TGF-β antibodies in the context of cancer is that theymay favor the appearance of new neoplastic lesions, due to theinhibition of the tumor-suppressive effect exerted by endogenous TGF-βon pre-cancerous cells.

One object of the invention is to provide a new strategy for improvingcancer treatment by targeting Tregs via their production of TGF-β.

It was previously shown that the production of TGF-β is tightlyregulated by a multi-step process. The precursor pro-TGF-β1homodimerizes prior to cleavage by pro-protein convertase FURIN. Theresulting product is called latent TGF-β1, in which the C-terminalfragment, or mature TGF-β1, remains non-covalently bound to theN-terminal fragment known as the Latency Associated Peptide, or LAP.This latent complex is inactive because LAP prevents mature TGF-β1 frombinding to its receptor.

In the present application, the inventors show that latent TGF-β isshown to bind to the surface of Tregs through the transmembrane proteinGARP (glycoprotein A repetitions predominant).

The present invention therefore provides a new strategy for targetingTreg based on an anti-GARP protein inhibiting TGF-β signaling.

SUMMARY

One object of the invention is a protein binding to Glycoprotein Arepetitions predominant (GARP) in the presence of TGF-β. In anembodiment, said protein binds to GARP only in the presence of TGF-β. Inanother embodiment, said protein binds to GARP when GARP is complexed toTGF-β. In another embodiment, said protein binds to a complex of GARPand TGF-β.

In an embodiment of the invention, said protein is an antibody moleculeselected from the group consisting of a whole antibody, a humanizedantibody, a single chain antibody, a dimeric single chain antibody, aFv, a Fab, a F(ab)′2, a defucosylated antibody, a bi-specific antibody,a diabody, a triabody, a tetrabody.

In another embodiment, said protein is an antibody fragment selectedfrom the group consisting of a unibody, a domain antibody, and ananobody.

In another embodiment, said protein is an antibody mimetic selected fromthe group consisting of an affibody, an affilin, an affitin, anadnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, afynomer, a versabody and a duocalin.

Another object of the invention is a protein as described here above ora protein binding GARP and inhibiting TGF-β signaling.

In an embodiment, said protein is an antibody or antigen bindingfragment thereof that binds to a conformational epitope comprising oneor more amino acids of GARP or an epitope of GARP modified as a resultof GARP being complexed with latent TGF-β.

In another embodiment, said antibody or antigen binding fragment thereoffurther binds one or more amino acids of latent TGF-β. In anotherembodiment, said antibody or antigen binding fragment thereof binds anepitope comprising one or more residues from residues 101 to 141 of GARPas set forth in SEQ ID NO: 1.

Another object of the invention is a protein having the variable regionof the heavy chain comprising at least one of the following CDRs:

(SEQ ID NO: 2) VH-CDR1: GFSLTGYGIN or (SEQ ID NO 52) GYGIN; (SEQ ID NO:3) VH-CDR2: MIWSDGSTDYNSVLTS; and (SEQ ID NO: 4) VH-CDR3: DRNYYDYDGAMDY,

-   -   or any CDR having an amino acid sequence that shares at least        60% identity with SEQ ID NO: 2-4 or 52,    -   or having the variable region of the light chain comprising at        least one of the following CDRs:

(SEQ ID NO: 5) VL-CDR1: KASDHIKNWLA; (SEQ ID NO: 6) VL-CDR2: GATSLEA;and (SEQ ID NO: 7) VL-CDR3: QQYWSTPWT,

-   -   or any CDR having an amino acid sequence that shares at least        60% identity with SEQ ID NO: 5-7;

or the variable region of the heavy chain comprises at least one of thefollowing CDRs:

(SEQ ID NO: 13) VH-CDR1: SYYID; (SEQ ID NO: 14) VH-CDR2:RIDPEDGGTKYAQKFQG; (SEQ ID NO: 15) VH-CDR3: or NEWETVVVGDLMYEYEY,

-   -   or any CDR having an amino acid sequence that shares at least        60% identity with SEQ ID NO: 13-15;    -   or wherein the variable region of the light chain comprises at        least one of the following CDRs:    -   VL-CDR1: QASQX₁I X₂S X₃LA (SEQ ID NO: 16), wherein X₁ is S or T,        X₂ is S or V, X₃ is Y or F;    -   VL-CDR2: X₁X₂SX₃X₄X₅T (SEQ ID NO: 17), wherein X₁ is G or R; X₂        is A or T; X₃ is R or I; X₄ is L or P; X₅ is Q or K;    -   VL-CDR3: QQYX₁SX₂PX₃T, wherein X₁ is D, A, Y or V; X₂ is A, L or        V; X₃ is V or P (SEQ ID NO: 18);    -   or any CDR having an amino acid sequence that shares at least        60% identity with SEQ ID NO: 16-18.

In an embodiment, the variable region of the heavy chain comprises atleast one of the following CDRs:

(SEQ ID NO: 2) VH-CDR1: GFSLTGYGIN or (SEQ ID NO: 52) GYGIN; (SEQ ID NO:3) VH-CDR2: MIWSDGSTDYNSVLTS; and (SEQ ID NO: 4) VH-CDR3: DRNYYDYDGAMDY,

-   -   or any CDR having an amino acid sequence that shares at least        60% identity with SEQ ID NO: 2-4 or 52,    -   and the variable region of the light chain comprises at least        one of the following CDRs:

(SEQ ID NO: 5) VL-CDR1: KASDHIKNWLA; (SEQ ID NO: 6) VL-CDR2: GATSLEA;and (SEQ ID NO: 7) VL-CDR3: QQYWSTPWT,

-   -   or any CDR having an amino acid sequence that shares at least        60% identity with SEQ ID NO: 5-7;

or the variable region of the heavy chain comprises at least one of thefollowing CDRs:

(SEQ ID NO: 13) VH-CDR1: SYYID; (SEQ ID NO: 14) VH-CDR2:RIDPEDGGTKYAQKFQG; (SEQ ID NO: 15) VH-CDR3: or NEWETVVVGDLMYEYEY;

-   -   or any CDR having an amino acid sequence that shares at least        60% identity with SEQ ID NO: 13-15,    -   and the variable region of the light chain comprises at least        one of the following CDRs:    -   VL-CDR1: QASQX₁I X₂S X₃LA (SEQ ID NO: 16), wherein X₁ is S or T,        X₂ is S or V, X₃ is Y or F;    -   VL-CDR2: X₁X₂SX₃X₄X₅T (SEQ ID NO: 17), wherein X₁ is G or R; X₂        is A or T; X₃ is R or I; X₄ is L or P; X₅ is Q or K;    -   VL-CDR3: QQYX₁SX₂PX₃T, wherein X₁ is D, A, Y or V; X₂ is A, L or        V; X₃ is V or P (SEQ ID NO: 18);    -   or any CDR having an amino acid sequence that shares at least        60% identity with SEQ ID NO: 16-18.

In another embodiment, the variable region of the heavy chain comprisesthe following CDRs: GFSLTGYGIN (SEQ ID NO: 2), MIWSDGSTDYNSVLTS (SEQ IDNO: 3), DRNYYDYDGAMDY (SEQ ID NO: 4) and the variable region of thelight chain comprises the following CDRs: KASDHIKNWLA (SEQ ID NO: 5),GATSLEA (SEQ ID NO: 6), QQYWSTPWT (SEQ ID NO: 7) or any CDR having anamino acid sequence that shares at least 60% identity with said SEQ IDNO: 2-7;

or the variable region of the heavy chain comprises the following CDRs:GYGIN (SEQ ID NO: 52), MIWSDGSTDYNSVLTS (SEQ ID NO: 3), DRNYYDYDGAMDY(SEQ ID NO: 4) and the variable region of the light chain comprises thefollowing CDRs: KASDHIKNWLA (SEQ ID NO: 5), GATSLEA (SEQ ID NO: 6),QQYWSTPWT (SEQ ID NO: 7) or any CDR having an amino acid sequence thatshares at least 60% identity with said SEQ ID NO: 52 and 3-7;

or wherein the variable region of the heavy chain comprises thefollowing CDRs: SYYID (SEQ ID NO: 13), RIDPEDGGTKYAQKFQG (SEQ ID NO:14), or NEWETVVVGDLMYEYEY (SEQ ID NO: 15); and the variable region ofthe light chain comprises the following CDRs: QASQX₁I X₂S X₃LA (SEQ IDNO: 16), wherein X₁ is S or T, X₂ is S or V, X₃ is Y or F; X₁X₂SX₃X₄X₅T(SEQ ID NO: 17), wherein X₁ is G or R; X₂ is A or T; X₃ is R or I; X₄ isL or P; X₅ is Q or K; QQYX₁SX₂PX₃T, wherein X₁ is D, A, Y or V; X₂ is A,L or V; X₃ is V or P (SEQ ID NO: 18); or any CDR having an amino acidsequence that shares at least 60% identity with said SEQ ID NO: 16-18.

In another embodiment, the amino acid sequence of the heavy chainvariable region is SEQ ID NO: 8 or SEQ ID NO: 50 and the amino acidsequence of the light chain variable region is SEQ ID NO: 9 or SEQ IDNO: 51, or the amino acid sequence of the heavy chain variable region isSEQ ID NO: 34 and the amino acid sequence of the light chain variableregion is one of SEQ ID NO: 35-39 or any sequence having an amino acidsequence that shares at least 60% identity with said SEQ ID NO: 8-9,50-51 or 34-39.

Another object of the invention is a protein as defined here abovebinding to an epitope on the polypeptide having the amino acid sequenceSEQ ID NO: 1 recognized by an antibody comprising a heavy chain variableregion as set forth in SEQ ID NO: 8 or in SEQ ID NO: 50 and a lightchain variable region as set forth in SEQ ID NO: 9 or in SEQ ID NO: 51,or by an antibody comprising a heavy chain variable region as set forthin SEQ ID NO: 34 and one of the light chain variable region as set forthin SEQ ID NO: 35-39.

Another object of the invention is an antibody or antigen bindingfragment produced by a hybridoma registered under the accession numberLMBP 10246CB on May 30, 2013.

Another object of the invention is a polynucleotide sequence encodingthe antibody or antigen binding fragment as described here above.

Another object of the invention is an expression vector comprising thepolynucleotide according to claim as described here above.

Another object of the invention is a hybridoma cell line producing anantibody against GARP registered under the accession number LMBP 10246CBon May 30, 2013.

Another object of the invention is a pharmaceutical compositioncomprising the protein as described here above and a pharmaceuticallyacceptable excipient.

Another object of the invention is a pharmaceutical composition asdescribed here above for treating a TGF-β related disorder in a subjectin need thereof. In an embodiment, the TGF-β related disorder isselected from the group consisting of inflammatory diseases, chronicinfection, cancer, fibrosis, cardiovascular diseases, cerebrovasculardisease (e.g. ischemic stroke), and neurodegenerative diseases.

In another embodiment, the pharmaceutical composition as described hereabove is to be administered in combination with another treatment forcancer or another immunotherapeutic agent such as a tumor vaccine or animmunostimulatory antibody.

In another embodiment, the pharmaceutical composition as described hereabove is to be administered as an immunostimulatory antibody fortreatment of cancer patients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. New monoclonal antibodies that recognize human GARP on the cellsurface. Murine BW5147 T cells, transfected or not with human GARP(hGARP) were stained with biotinylated in-house anti-hGARP antibodies(MHGARP1 to 9) and streptavidin-PE (SA-PE, top panels), or with acommercial anti-hGARP antibody (clone Plato-1) and secondary anti-mouseIgG2b coupled to AlexaFluor 488 (AF488, bottom panels).

FIGS. 2A-2B. MHGARP8 inhibits active TGF-β production by a human Tregclone. Clone Treg A1 was stimulated during 24 hours with >CD3/CD28antibodies, alone or in the presence of the indicated anti-hGARP mAbs(20 μg/ml). (FIG. 2A) Cell lysates were analyzed by WB with anti-pSMAD2and anti-β-ACTIN antibodies. (FIG. 2B) Quantification of ECL signalsfrom WB shown in A.

FIGS. 3A-3E. (FIG. 3A) Regions in the hGARP protein required for bindingby anti-hGARP antibodies. Murine BW5147 T cells expressing the HA-taggedproteins schematized on the left were stained with anti-hGARP (MHGARP1to 9, as indicated on top of the figure) or anti-HA antibodies, andanalyzed by flow cytometry. Histograms are gated on live cells. Based onthe FACS results, regions required for binding by the various MHGARPmAbs were identified and are indicated by horizontal bars above therepresentations of the HA-tagged chimeras.

(FIG. 3B) Abundance of the epitope recognized by MHGARP-8 increases uponoverexpression of TGF-β1. Parental BW5147 T cells (BW untransfected) orclones stably transfected with hGARP alone (BW+hGARP) or with hTGFB1(BW+hGARP+hTGF-b1) were stained as in A, or with anti-mLAP-AF647 oranti-hLAP-APC antibodies, and analyzed by flow cytometry.

(FIG. 3C) MHGARP-1, -2, -3, -4 and -5 recognize free hGARP, but nothGARP bound to TGF-β1. Cell lysates from parental BW5147 T cells or aclone stably transfected with hGARP and hTGFB1 were imunoprecipitatedwith anti-hGARP mAbs (MHGARP1 to 9, as indicated on top of the figure).Cell lysates (30% input) or IP products were analyzed by Western blotwith a commercial anti-hGARP mAb (clone Plato-1, top panels) and with anantibody directed against a C-terminal epitope of TGF-β1, which detectspro-TGF-β1 as a 50 kDa band and mature TGF-β1 as a 13 kDa band (bottompanels). * Aspecific product detected in untransfected cells.

(FIG. 3D) Overexpression of hTGFB1 in hGARP-transfected 293T cellsdecreases binding of MHGARP-1, -2, -3, -4, and -5, but increases bindingof MHGARP-8. 293T cells were co-transfected with a hGARP-encodingplasmid (0.25 rig), the indicated amounts of a hTGFB1-encoding plasmid,and an empty plasmid to bring the total amount of transfected DNA to 2.5μg in all conditions. Transfected cells were stained with anti-hGARPmAbs (MHGARP1 to 9, as indicated on top of the figure), and analyzed byflow cytometry.

(FIG. 3E) Silencing of hTGFB1 in hGARP-transduced JURKAT cells decreasesbinding of MHGARP-8. JURKAT cells, transduced or not with hGARP, weretransfected with siRNA specific for the TGFB1 mRNA (siTGFB1) or ascramble siRNA control. Transfected cells were stained with anti-hGARPmAbs (MHGARP1 to 9, as indicated on top of the figure) or with ananti-hLAP antibody, and analyzed by flow cytometry.

FIGS. 4A-4B. Presentation of hTGF-β1 on the cell surface is notsufficient for binding by MHGARP8. 293T cells were transfected asindicated below, stained with anti-hLAP antibodies or with MGARP8, thenanalyzed by flow cytometry.

(FIG. 4A) Transfection with constructs encoding the HA-tagged proteinsschematized on the left, without a hTGFB1 construct.

(FIG. 4B) Co-transfection with constructs encoding the HA-taggedproteins schematized on the left, with a hTGFB1 construct.

FIG. 5. Binding of MHGARP-2, -3 and -8 requires amino-acids 137-138-139of hGARP. Parental BW5147 T cells (BW untransfected) or clones stablytransfected with plasmids encoding HA-tagged forms of hGARP were stainedwith the indicated anti-hGARP or anti-HA antibodies, and analyzed byflow cytometry. The HA-tagged forms of hGARP tested here comprised aa20-662 of hGARP (wild type, WT), or aa 20-662 of hGARP in which groupsof 3 amino-acids located in region 101-141 were replaced by theamino-acids found in the corresponding region of mGARP (Mut I, Mut IIand Mut III). Amino-acid sequences of region 101-141 of hGARP—WT (SEQ IDNO: 62), -Mut I (SEQ ID NO: 63), -Mut II (SEQ ID NO: 64), -Mut III (SEQID NO: 65) and mGARP (SEQ ID NO: 66) are indicated on the left.Amino-acids that differ between human and mouse GARP are highlighted bygrey vertical boxes, and amino-acids mutated in Mut I, Mut II and MutIII are indicated by black horizontal boxes.

FIGS. 6A-6B. MHGARP8 inhibits Treg function in vivo. On day 0, theindicated groups of NSG mice received i.v. injections of human PBMCs, incombination or not with human Tregs. Mice from groups III and IV weretreated with the MHGARP8 antibody, injected i.p. once a week, startingon day −1. Objective signs of GvHD development in the recipient micewere monitored bi-weekly. A GvHD score was established based on weightloss (0: <10%; 1: 10%-20%; 2: >20%; 3: >30%), anemia (0: red or pinktail; 1: white tail), posture (0: normal; 1: hump), general activity (0:normal; 1: limited), hair loss (0: no hair loss; 1: hair loss) andicterus (0: white or red tail; 1: yellow tail). Maximum disease severityor death corresponded to a score of 7. (FIG. 6A) Experiment 1. Valuesrepresent mean scores. (FIG. 6B) Experiment 2. Values represent meanscores+SEM.

FIGS. 7A-7B. New anti-hGARP mAbs. (FIG. 7A) Schematic representation ofthe experimental strategies used to derive anti-hGARP mAbs. (FIG. 7B)Flow cytometry analyses of clone ThA2 (human CD4+Th cells which do notexpress hGARP), or ThA2 cells transduced with hGARP, after staining withbiotinylated MHG-1 to -14 mAbs and streptavidin coupled to PE (SA-PE),with LHG-1 to -17 mAbs and a secondary anti-hIgG1 antibody coupled toPE, or with a commercially available mouse anti-hGARP mAb (clonePlato-1) and a secondary anti-mIgG2b antibody coupled to AF647.

FIGS. 8A-8B. Immune responses from immunized llamas. (FIG. 8A) showsimmune responses from DNA immunized llamas. (FIG. 8B) shows immuneresponses from llamas immunized with BW cells expressing hGARP/hTGFβ.

FIG. 9. Cross-reactivity to cynomolgus GARP-TGFβ measured on cells byFACS. 293E cells were transfected with human/cyno GARP and human/cynoTGFB. LHG-10-D and the affinity optimized variants are cross-reactivewith cynomolgus GARP-TGFB.

FIG. 10. Sequences of LHG-10 antibodies and its shuffle variants. FIG.10 discloses SEQ ID NOS: 34, 35, 35, 37, 39, 36, 38, 35, 37, 39, 36, 38,13-15 and 19-33, respectively, in order of appearance.

FIGS. 11A-11B. MHGARP8 and LHG-10 inhibit production of active TGF-β byhuman Tregs. After a short in vitro amplification, humanCD4+CD25hiCD127lo cells (Tregs) were re-stimulated with anti-CD3/CD28coated beads during 24 hours, in the presence or absence of theindicated mAbs (10 μg/ml). Cells lysates were analyzed by Western Blotwith antibodies against phosphorylated SMAD2 (pSMAD2), as a read-out foractive TGF-β production, or β-ACTIN (loading control).

FIGS. 12A-12B. MHGARP8 and LHG-10 inhibit the suppressive activity ofhuman Tregs in vitro. (FIG. 12A) Freshly isolated human CD4+CD25−CD127hicells (Th; 2×10⁴ per microwell) were seeded alone or with clone Treg A1(Stockis, J. et al. Eur. J. Immunol. 2009, 39:869-882) at a 1/1 Treg toTh ratio. Cells were stimulated with coated anti-CD3 and solubleanti-CD28, in the presence or absence of the indicated anti-hGARP mAbs(10 μg/ml). 3H-Thymidine (3H-Thy) was added during the last 16 hours ofa 4-day culture and incorporation was measured in a scintillationcounter as a read-out for proliferation. Bar histograms indicate kcpm(means of triplicates+SD). Clone Treg A1 did not proliferate in theabsence of Th cells (Treg alone: 0.5±0.04 kcpm). Suppression of Thproliferation in the presence of Tregs is indicated above each blackbar, and is calculated as follows: % suppression=1−(kcpm (Th alone)/kcpm(Th+Treg). (FIG. 12B) Clone ThA2 cells (Th; 1×10⁴ per microwell) wereseeded with clone Treg A1 at the indicated Treg to Th ratios, in thepresence or absence of MHGARP8 (MHG-8), anti-hTGF-β1 mAb (clone 1D11) oran isotype control (mIgG1). Stimulation, measure of proliferation andcalculation of suppression were performed as in A.

FIGS. 13A-13B. Forms and regions of GARP bound by anti-GARP mAbs. (FIG.13A) Schematic representations of GARP and GARP/TGF-β complexes. ProteinGARP is represented by a thick curved grey line. Numbers indicateamino-acid positions. TGF-β is represented with the Latency AssociatedPeptide (LAP) as thick black lines, and the mature TGF-β1 peptide asthick straight grey lines. Thin black lines represent inter-chaindisulfide bonds. (FIG. 13B) Classification of anti-hGARP mAbs based ontheir binding requirements.

FIGS. 14A-14B. Three groups of anti-hGARP mAbs bind free GARP only, freeGARP and GARP/TGF-β1 complexes, or GARP/TGF-β1 complexes only,respectively. (FIG. 14A) Cell lysates of BW cells transfected with hGARPand hTGFB1 were immunoprecipitated with the indicated anti-hGARP mAbs.Total lysates (BW+hGARP+hTGFB1 or untransfected controls) and IPproducts were analysed by Western Blot with antibodies against hGARP(clone Plato-1), LAP or the mature TGF-β peptide. (FIG. 14B) Flowcytometry analyses of 293T cells, untransfected or transfected withhGARP, hTGFB1 or both, and stained as indicated with anti-LAP-APC,biotinylated MHG mAbs and streptavidin-PE, clone Plato-1 andanti-mIgG2b-AF647, or LHG mAbs and anti-hIgG1-PE.

FIGS. 15A-15B. Amino-acids of hGARP required for binding by MHG and LHGmAbs. (FIG. 15A) Flow cytometry analyses of 293T cells transfected withplasmids encoding the HA-tagged mGARP/hGARP chimeras schematized on theleft (numbers represent amino-acid positions in hGARP). Cells werestained with biotinylated MHG mAbs and strepatvidin-PE, LHG mAbs andanti-hIgG1-PE, or anti-HA and anti-mIgG1-AF647. hTGFB1 wasco-transfected with mGARP/hGARP chimeras for the analyses of mAbs thatbind hGARP/hTGF-β1 complexes only (LHG-3, MHGARP8 (MHG-8), LHG-10).(FIG. 15B) As above, except that 293T cells were transfected withplasmids encoding mutated forms of full-length HA-tagged hGARP. In eachmutant, 3 amino-acids of hGARP were replaced by the 3 amino-acids foundin mGARP, as illustrated in the alignment on the left (numbers representamino-acid positions in hGARP). FIG. 15B discloses SEQ ID NOS: 62-66,respectively, in order of appearance.

FIGS. 16A-16C. Inhibition of human Treg function by anti-hGARP in vivo.(FIG. 16A) shows the protocol on day 0, the indicated groups of NSG micereceived i.v. injections of human PBMCs, in combination or not withhuman Tregs. (FIG. 16B) shows the results of 4 independent experiments(I to IV), performed with cells from donors A, B or C, with theindicated numbers of mice per group (n). Disease onset is the day whenmean disease score becomes >1, and is indicated for 3 experimentalgroups in which mice were grafted with PBMCs only (group a), PBMCs andTregs (group b), or PBMCs and Tregs and treated with MHGARP8 (MHG-8)(group c). (FIG. 16C) Detailed results from experiment IV, showing theevolution of mean disease score (left) and survival curves (right) inthe indicated groups of mice. Statistical significance of differencesbetween groups b (PBMCs+Tregs) and c (PBMCs+Tregs+MHG-8) were calculatedusing 2-way Anova analysis for progression of disease scores (p=0.0001),and a Log-rank (Mantel-Cox) test for survival (p=0.0027).

FIG. 17. Anti-hGARP mAbs that block TGF-β production inhibit suppressionby human Tregs in vivo. Experiment performed according to protocol shownin FIG. 16A. Top graph shows progression of disease scores (means pergroup+sem), bottom graph shows survival.

FIGS. 18A-18C. Inhibition of human Tregs by MHG-8 increases cytokineproduction and T cell proliferation in vivo.

NSG mice injected as in FIG. 16A were sacrificed 20 days after celltransfer. (FIG. 18A) Serum levels of human cytokines were measured in amultiplex bead assay. (FIGS. 18B-18C) Numbers and proportions of theindicated human cells in the spleens were evaluated by flow cytometry.Squares and triangles represent individual mice (N=4-5 mice per group).Circles represent numbers or proportions of the corresponding humancells injected per mouse on day 0. Statistical significance betweengroups was determined with a Student's t-test. P values are indicatedabove brackets. Data from one experiment representative of two. (FIG.18D) Post-graft evaluation of the proportion and number of GARP⁺ Tregsand number of activated Tregs following anti-GARP mAb treatment.

FIGS. 19A-19D. Impact of mutations in GARP or TGF-β1 on the binding ofanti-GARP or anti-LAP antibodies to GARP/TGF-β1 complexes.

(FIG. 19A) Mean percent residual binding of MHG-8 to GARP/TGF-β1complexes containing the mutations indicated below the bar graphs. (FIG.19B) Mean percent residual binding of LHG-10 to GARP/TGF-β1 complexescontaining the mutations indicated below the bar graphs. (FIG. 19C) Meanpercent residual binding of MHG-6 to GARP/TGF-β1 complexes containingthe mutations indicated below the bar graphs. Ratios of EC50 were notmeasured for MHG-6. (FIG. 19D) Mean percent residual binding of ananti-LAP antibody. Ratios of EC50 were not measured for anti-LAP toGARP/TGF-β1 complexes containing the mutations indicated below the bargraphs. n: number of independent experiments. The number indicated beloweach mutation corresponds to the ratio between the EC50 of MHG-8 bindingto the mutant and the EC50 of MHG-8 binding to the WT. Mutations with aratio >2 are considered to decrease the avidity for binding by MHG-8.NE: non evaluable (residual binding <10%); nt: not tested.

FIGS. 20A-20C. Impact of mutations in GARP or TGF-β1 on the inhibitoryactivity of MHG-8 and LHG-10.

(FIG. 20A) Residual inhibitory activity of MHG-8 on the indicatedmutated forms of GARP or TGF-β1 (means of triplicates). Mutations thatare underlined induced complete loss of binding by MHG-8, as illustratedin FIG. 19A. (FIG. 20B) Residual inhibitory activity of LHG-10 on theindicated mutated forms of GARP or TGF-β1 (means of triplicates).Mutations that are underlined induced complete loss of binding byLHG-10, as illustrated in FIG. 19B. (FIG. 20C) Residual inhibitoryactivity of the control anti-TGF-β1 neutralizing antibody on theindicated mutated forms of GARP or TGF-β1 (means of triplicates). Errorbars correspond to standard deviation. Negative values were brought tozero.

DETAILED DESCRIPTION Definitions

In the present invention, the following terms have the followingmeanings: “Antibody” or “Immunoglobulin”—As used herein, the term“immunoglobulin” includes a polypeptide having a combination of twoheavy and two light chains whether or not it possesses any relevantspecific immunoreactivity. “Antibodies” refers to such assemblies whichhave significant known specific immunoreactive activity to an antigen ofinterest (e.g. human GARP). The term “GARP antibodies” is used herein torefer to antibodies which exhibit immunological specificity for humanGARP protein. As explained elsewhere herein, “specificity” for humanGARP does not exclude cross-reaction with species homologues of GARP. Inaddition, it also does not exclude antibodies recognising an epitopespanning GARP protein residues and TGF-β protein residue. Antibodies andimmunoglobulins comprise light and heavy chains, with or without aninterchain covalent linkage between them. Basic immunoglobulinstructures in vertebrate systems are relatively well understood. Thegeneric term “immunoglobulin” comprises five distinct classes ofantibody that can be distinguished biochemically. All five classes ofantibodies are within the scope of the present invention, the followingdiscussion will generally be directed to the IgG class of immunoglobulinmolecules. With regard to IgG, immunoglobulins comprise two identicallight polypeptide chains of molecular weight approximately 23,000Daltons, and two identical heavy chains of molecular weight53,000-70,000 Daltons. The four chains are joined by disulfide bonds ina “Y” configuration wherein the light chains bracket the heavy chainsstarting at the mouth of the “Y” and continuing through the variableregion. The light chains of an antibody are classified as either kappaor lambda ([κ], [λ]). Each heavy chain class may be bonded with either akappa or lambda light chain. In general, the light and heavy chains arecovalently bonded to each other, and the “tail” regions of the two heavychains are bonded to each other by covalent disulfide linkages ornon-covalent linkages when the immunoglobulins are generated either byhybridomas, B cells or genetically engineered host cells. In the heavychain, the amino acid sequences run from an N-terminus at the forkedends of the Y configuration to the C-terminus at the bottom of eachchain. Those skilled in the art will appreciate that heavy chains areclassified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) withsome subclasses among them (e.g., γ1-γ4). It is the nature of this chainthat determines the “class” of the antibody as IgG, IgM, IgA IgG, orIgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1,IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known toconfer functional specialization. Modified versions of each of theseclasses and isotypes are readily discernable to the skilled artisan inview of the instant disclosure and, accordingly, are within the scope ofthe instant invention. As indicated above, the variable region of anantibody allows the antibody to selectively recognize and specificallybind epitopes on antigens. That is, the VL domain and VH domain of anantibody combine to form the variable region that defines a threedimensional antigen binding site. This quaternary antibody structureforms the antigen binding site present at the end of each arm of the Y.More specifically, the antigen binding site is defined by threecomplementarity determining regions (CDRs) on each of the VH and VLchains.

“An isolated antibody”—As used herein, an “isolated antibody” is onethat has been separated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials that would interfere with diagnostic or therapeutic uses ofthe antibody, and may include enzymes, hormones, and other proteinaceousor non proteinaceous components. In preferred embodiments, the antibodyis purified: (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight; (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator; or (3)to homogeneity as shown by SDS-PAGE under reducing or non-reducingconditions and using Coomassie blue or, preferably, silver staining. Anisolated antibody includes the antibody in situ within recombinant cellssince at least one component of the antibody's natural environment willnot be present. Ordinarily, however, an isolated antibody will beprepared by at least one purification step.

“Affinity variants”—As used herein, the term “affinity variant” refersto a variant antibody which exhibits one or more changes in amino acidsequence compared to a reference GARP antibody, wherein the affinityvariant exhibits an altered affinity for the human GARP protein orGARP/TGF-β complex in comparison to the reference antibody. Typically,affinity variants will exhibit an improved affinity for human GARP orhuman GARP/TGF-β complex, as compared to the reference GARP antibody.The improvement may be a lower KD for human GARP, a faster off-rate forhuman GARP, or an alteration in the pattern of cross-reactivity withnon-human GARP homologues. Affinity variants typically exhibit one ormore changes in amino acid sequence in the CDRs, as compared to thereference GARP antibody. Such substitutions may result in replacement ofthe original amino acid present at a given position in the CDRs with adifferent amino acid residue, which may be a naturally occurring aminoacid residue or a non-naturally occurring amino acid residue. The aminoacid substitutions may be conservative or non-conservative.

“Binding Site”—As used herein, the term “binding site” comprises aregion of a polypeptide which is responsible for selectively binding toa target antigen of interest (e.g. human GARP). Binding domains orbinding regions comprise at least one binding site. Exemplary bindingdomains include an antibody variable domain. The antibody molecules ofthe invention may comprise a single antigen binding site or multiple(e.g., two, three or four) antigen binding sites.

“Conservative amino acid substitution”—As used herein, a “conservativeamino acid substitution” is one in which the amino acid residue isreplaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in an immunoglobulin polypeptide may be replaced with anotheramino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members.

“Chimeric”—As used herein, a “chimeric” protein comprises a first aminoacid sequence linked to a second amino acid sequence with which it isnot naturally linked in nature. The amino acid sequences may normallyexist in separate proteins that are brought together in the fusionpolypeptide or they may normally exist in the same protein but areplaced in a new arrangement in the fusion polypeptide. A chimericprotein may be created, for example, by chemical synthesis, or bycreating and translating a polynucleotide in which the peptide regionsare encoded in the desired relationship. Exemplary chimeric GARPantibodies include fusion proteins comprising camelid-derived VH and VLdomains, or humanised variants thereof, fused to the constant domains ofa human antibody, e.g. human IgG1, IgG2, IgG3 or IgG4.

“CDR”—As used herein, the term “CDR” or “complementarity determiningregion” means the non-contiguous antigen combining sites found withinthe variable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), by Chothia et al., J. Mol. Biol.196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745(1996) where the definitions include overlapping or subsets of aminoacid residues when compared against each other. The amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth for comparison. Preferably, the term “CDR” is aCDR as defined by Kabat based on sequence comparisons.

TABLE 1 CDR definitions CDR definitions Kabat (1) Chothia (2) MacCallum(3) VH CDR1 31-35 26-32 30-35 VH CDR2 50-65 53-55 47-58 VH CDR3  95-102 96-101  93-101 VL CDR1 24-34 26-32 30-36 VL CDR2 50-56 50-52 46-55 VLCDR3 89-97 91-96 89-96 (1) Residue numbering follows the nomenclature ofKabat et al., supra (2) Residue numbering follows the nomenclature ofChothia et al., supra (3) Residue numbering follows the nomenclature ofMacCallum et al., supra

“CH2 domain”—As used herein the term “CH2 domain” includes the region ofa heavy chain molecule that extends, e.g., from about residue 244 toresidue 360 of an antibody using conventional numbering schemes(residues 244 to 360, Kabat numbering system; and residues 231-340, EUnumbering system, Kabat E A et al. Sequences of Proteins ofImmunological Interest. Bethesda, US Department of Health and HumanServices, NIH. 1991). The CH2 domain is unique in that it is not closelypaired with another domain. Rather, two N-linked branched carbohydratechains are interposed between the two CH2 domains of an intact nativeIgG molecule. It is also well documented that the CH3 domain extendsfrom the CH2 domain to the C-terminal of the IgG molecule and comprisesapproximately 108 residues.

“Camelid-Derived”—In certain preferred embodiments, the GARP antibodymolecules of the invention comprise framework amino acid sequencesand/or CDR amino acid sequences derived from a camelid conventionalantibody raised by active immunization of a camelid with GARP antigen.However, GARP antibodies comprising camelid-derived amino acid sequencesmay be engineered to comprise framework and/or constant region sequencesderived from a human amino acid sequence or other non-camelid mammalianspecies. For example, a human or non-human primate framework region,heavy chain region, and/or hinge region may be included in the subjectGARP antibodies. In an embodiment, one or more non-camelid amino acidsmay be present in the framework region of a “camelid-derived” GARPantibody, e.g., a camelid framework amino acid sequence may comprise oneor more amino acid mutations in which the corresponding human ornon-human primate amino acid residue is present. Moreover,camelid-derived VH and VL domains, or humanized variants thereof, may belinked to the constant domains of human antibodies to produce a chimericmolecule, as extensively described elsewhere herein.

“Derived From”—As used herein the term “derived from” a designatedprotein (e.g. a GARP antibody or antigen-binding fragment thereof)refers to the origin of the polypeptide. In an embodiment, thepolypeptide or amino acid sequence which is derived from a particularstarting polypeptide is a CDR sequence or sequence related thereto. Inan embodiment, the amino acid sequence which is derived from aparticular starting polypeptide is not contiguous. For example, in anembodiment, one, two, three, four, five, or six CDRs are derived from astarting antibody. In an embodiment, the polypeptide or amino acidsequence which is derived from a particular starting polypeptide oramino acid sequence has an amino acid sequence that is essentiallyidentical to that of the starting sequence, or a region thereof whereinthe region consists of at least of at least 3-5 amino acids, 5-10 aminoacids, at least 10-20 amino acids, at least 20-30 amino acids, or atleast 30-50 amino acids, or which is otherwise identifiable to one ofordinary skill in the art as having its origin in the starting sequence.In an embodiment, the one or more CDR sequences derived from thestarting antibody are altered to produce variant CDR sequences, e.g.affinity variants, wherein the variant CDR sequences maintain GARPbinding activity.

“Diabodies”—As used herein, the term “diabodies” refers to smallantibody fragments prepared by constructing sFv fragments (see sFvparagraph) with short linkers (about 5-10 residues) between the VH andVL domains such that inter-chain but not intra-chain pairing of the Vdomains is achieved, resulting in a bivalent fragment, i.e., fragmenthaving two antigen-binding sites. Bispecific diabodies are heterodimersof two “crossover” sFv fragments in which the VH and VL domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHolliger et al., Proc. Natl. Acad. Sci., 90:6444-6448 (1993).

“Engineered”—As used herein the term “engineered” includes manipulationof nucleic acid or polypeptide molecules by synthetic means (e.g. byrecombinant techniques, in vitro peptide synthesis, by enzymatic orchemical coupling of peptides or some combination of these techniques).Preferably, the antibodies of the invention are engineered, includingfor example, humanized and/or chimeric antibodies, and antibodies whichhave been engineered to improve one or more properties, such as antigenbinding, stability/half-life or effector function.

“Epitope”—As used herein, the term “epitope” refers to a specificarrangement of amino acids located on a peptide or protein or proteinsto which an antibody binds. Epitopes often consist of a chemicallyactive surface grouping of molecules such as amino acids or sugar sidechains, and have specific three dimensional structural characteristicsas well as specific charge characteristics. Epitopes can be linear orconformational, i.e., involving two or more sequences of amino acids invarious regions of the antigen that may not necessarily be contiguous.

“Framework region”—The term “framework region” or “FR region” as usedherein, includes the amino acid residues that are part of the variableregion, but are not part of the CDRs (e.g., using the Kabat definitionof CDRs). Therefore, a variable region framework is between about100-120 amino acids in length but includes only those amino acidsoutside of the CDRs. For the specific example of a heavy chain variableregion and for the CDRs as defined by Kabat et al., framework region 1corresponds to the domain of the variable region encompassing aminoacids 1-30; framework region 2 corresponds to the domain of the variableregion encompassing amino acids 36-49; framework region 3 corresponds tothe domain of the variable region encompassing amino acids 66-94, andframework region 4 corresponds to the domain of the variable region fromamino acids 103 to the end of the variable region. The framework regionsfor the light chain are similarly separated by each of the light claimvariable region CDRs. Similarly, using the definition of CDRs by Chothiaet al. or McCallum et al. the framework region boundaries are separatedby the respective CDR termini as described above. In preferredembodiments the CDRs are as defined by Kabat. In naturally occurringantibodies, the six CDRs present on each monomeric antibody are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding site as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe heavy and light variable domains show less inter-molecularvariability in amino acid sequence and are termed the framework regions.The framework regions largely adopt a [beta]-sheet conformation and theCDRs form loops which connect, and in some cases form part of, the[beta]-sheet structure. Thus, these framework regions act to form ascaffold that provides for positioning the six CDRs in correctorientation by inter-chain, non-covalent interactions. The antigenbinding site formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto the immunoreactive antigen epitope. The position of CDRs can bereadily identified by one of ordinary skill in the art.

“Fragment”—As used herein, the term “fragment” refers to a part orregion of an antibody or antibody chain comprising fewer amino acidresidues than an intact or complete antibody or antibody chain. The term“antigen-binding fragment” refers to a polypeptide fragment of animmunoglobulin or antibody that binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding to human GARP). As usedherein, the term “fragment” of an antibody molecule includesantigen-binding fragments of antibodies, for example, an antibody lightchain variable domain (VL), an antibody heavy chain variable domain(VH), a single chain antibody (scFv), a F(ab′)2 fragment, a Fabfragment, an Fd fragment, an Fv fragment, a single domain antibodyfragment (DAb), a one-armed (monovalent) antibody, diabodies or anyantigen-binding molecule formed by combination, assembly or conjugationof such antigen binding fragments. Fragments can be obtained, e.g., viachemical or enzymatic treatment of an intact or complete antibody orantibody chain or by recombinant means.

“Fv”—As used herein, the term “Fv” is the minimum antibody fragment thatcontains a complete antigen-recognition and -binding site. This fragmentconsists of a dimer of one heavy- and one light-chain variable regiondomain in tight, non-covalent association. From the folding of these twodomains emanate six hypervariable loops (three loops each from the H andL chain) that contribute the amino acid residues for antigen binding andconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

“Heavy chain region”—As used herein, the term “heavy chain region”includes amino acid sequences derived from the constant domains of animmunoglobulin heavy chain. A polypeptide comprising a heavy chainregion comprises at least one of: a CH1 domain, a hinge (e.g., upper,middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain,or a variant or fragment thereof. In an embodiment, a binding moleculeof the invention may comprise the Fc region of an immunoglobulin heavychain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). Inanother embodiment, a binding molecule of the invention lacks at least aregion of a constant domain (e.g., all or part of a CH2 domain). Incertain embodiments, at least one, and preferably all, of the constantdomains are derived from a human immunoglobulin heavy chain. Forexample, in one preferred embodiment, the heavy chain region comprises afully human hinge domain. In other preferred embodiments, the heavychain region comprising a fully human Fc region (e.g., hinge, CH2 andCH3 domain sequences from a human immunoglobulin). In certainembodiments, the constituent constant domains of the heavy chain regionare from different immunoglobulin molecules. For example, a heavy chainregion of a polypeptide may comprise a CH2 domain derived from an IgG1molecule and a hinge region derived from an IgG3 or IgG4 molecule. Inother embodiments, the constant domains are chimeric domains comprisingregions of different immunoglobulin molecules. For example, a hinge maycomprise a first region from an IgG1 molecule and a second region froman IgG3 or IgG4 molecule. As set forth above, it will be understood byone of ordinary skill in the art that the constant domains of the heavychain region may be modified such that they vary in amino acid sequencefrom the naturally occurring (wild-type) immunoglobulin molecule. Thatis, the polypeptides of the invention disclosed herein may comprisealterations or modifications to one or more of the heavy chain constantdomains (CH1, hinge, CH2 or CH3) and/or to the light chain constantdomain (CL). Exemplary modifications include additions, deletions orsubstitutions of one or more amino acids in one or more domains.

“Hinge region”—As used herein, the term “hinge region” includes theregion of a heavy chain molecule that joins the CH1 domain to the CH2domain. This hinge region comprises approximately 25 residues and isflexible, thus allowing the two N-terminal antigen binding regions tomove independently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al. J. Immunol.1998 161:4083).

The terms “hypervariable loop” and “complementarity determining region”are not strictly synonymous, since the hypervariable loops (HVs) aredefined on the basis of structure, whereas complementarity determiningregions (CDRs) are defined based on sequence variability (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1983) and thelimits of the HVs and the CDRs may be different in some VH and VLdomains. The CDRs of the VL and VH domains can typically be defined ascomprising the following amino acids: residues 24-34 (CDRL1), 50-56(CDRL2) and 89-97 (CDRL3) in the light chain variable domain, andresidues 31-35 or 31-35b (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) inthe heavy chain variable domain; (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). Thus, the HVs may becomprised within the corresponding CDRs and references herein to the“hypervariable loops” of VH and VL domains should be interpreted as alsoencompassing the corresponding CDRs, and vice versa, unless otherwiseindicated. The more highly conserved regions of variable domains arecalled the framework region (FR), as defined below. The variable domainsof native heavy and light chains each comprise four FRs (FR1, FR2, FR3and FR4, respectively), largely adopting a [beta]-sheet configuration,connected by the three hypervariable loops. The hypervariable loops ineach chain are held together in close proximity by the FRs and, with thehypervariable loops from the other chain, contribute to the formation ofthe antigen-binding site of antibodies. Structural analysis ofantibodies revealed the relationship between the sequence and the shapeof the binding site formed by the complementarity determining regions(Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al.,J. Mol. Biol, 215: 175-182 (1990)). Despite their high sequencevariability, five of the six loops adopt just a small repertoire ofmain-chain conformations, called “canonical structures”. Theseconformations are first of all determined by the length of the loops andsecondly by the presence of key residues at certain positions in theloops and in the framework regions that determine the conformationthrough their packing, hydrogen bonding or the ability to assume unusualmain-chain conformations.

“Humanising substitutions”—As used herein, the term “humanisingsubstitutions” refers to amino acid substitutions in which the aminoacid residue present at a particular position in the VH or VL domainantibody GARP antibody (for example a camelid-derived GARP antibody) isreplaced with an amino acid residue which occurs at an equivalentposition in a reference human VH or VL domain. The reference human VH orVL domain may be a VH or VL domain encoded by the human germline, inwhich case the substituted residues may be referred to as “germliningsubstitutions”. Humanising/germlining substitutions may be made in theframework regions and/or the CDRs of a GARP antibody, defined herein.

“High human homology”—An antibody comprising a heavy chain variabledomain (VH) and a light chain variable domain (VL) will be considered ashaving high human homology if the VH domains and the VL domains, takentogether, exhibit at least 90% amino acid sequence identity to theclosest matching human germline VH and VL sequences. Antibodies havinghigh human homology may include antibodies comprising VH and VL domainsof native non-human antibodies which exhibit sufficiently high %sequence identity human germline sequences, including for exampleantibodies comprising VH and VL domains of camelid conventionalantibodies, as well as engineered, especially humanized, variants ofsuch antibodies and also “fully human” antibodies. In an embodiment theVH domain of the antibody with high human homology may exhibit an aminoacid sequence identity or sequence homology of 80% or greater with oneor more human VH domains across the framework regions FR1, FR2, FR3 andFR4. In other embodiments the amino acid sequence identity or sequencehomology between the VH domain of the polypeptide of the invention andthe closest matching human germline VH domain sequence may be 85% orgreater, 90% or greater, 95% or greater, 97% or greater, or up to 99% oreven 100%. In an embodiment the VH domain of the antibody with highhuman homology may contain one or more (e.g. 1 to 10) amino acidsequence mis-matches across the framework regions FR1, FR2, FR3 and FR4,in comparison to the closest matched human VH sequence. In anotherembodiment the VL domain of the antibody with high human homology mayexhibit a sequence identity or sequence homology of 80% or greater withone or more human VL domains across the framework regions FR1, FR2, FR3and FR4. In other embodiments the amino acid sequence identity orsequence homology between the VL domain of the polypeptide of theinvention and the closest matching human germline VL domain sequence maybe 85% or greater 90% or greater, 95% or greater, 97% or greater, or upto 99% or even 100%.

In an embodiment the VL domain of the antibody with high human homologymay contain one or more (e.g. 1 to 10) amino acid sequence mis-matchesacross the framework regions FR1, FR2, FR3 and FR4, in comparison to theclosest matched human VL sequence. Before analyzing the percentagesequence identity between the antibody with high human homology andhuman germline VH and VL, the canonical folds may be determined, whichallow the identification of the family of human germline segments withthe identical combination of canonical folds for H1 and H2 or L1 and L2(and L3). Subsequently the human germline family member that has thehighest degree of sequence homology with the variable region of theantibody of interest is chosen for scoring the sequence homology. Thedetermination of Chothia canonical classes of hypervariable loops L1,L2, L3, H1 and H2 can be performed with the bioinformatics toolspublicly available on webpage www.bioinf.org.uk/abs/chothia.html.page.The output of the program shows the key residue requirements in a datafile. In these data files, the key residue positions are shown with theallowed amino acids at each position. The sequence of the variableregion of the antibody of interest is given as input and is firstaligned with a consensus antibody sequence to assign the Kabat numberingscheme. The analysis of the canonical folds uses a set of key residuetemplates derived by an automated method developed by Martin andThornton (Martin et al., J. Mol. Biol. 263:800-815 (1996)). With theparticular human germline V segment known, which uses the samecombination of canonical folds for H1 and H2 or L1 and L2 (and L3), thebest matching family member in terms of sequence homology can bedetermined. With bioinformatics tools the percentage sequence identitybetween the VH and VL domain framework amino acid sequences of theantibody of interest and corresponding sequences encoded by the humangermline can be determined, but actually manual alignment of thesequences can be applied as well. Human immunoglobulin sequences can beidentified from several protein data bases, such as VBase(http://vbase.mrc-cpe.cam.ac.uk/) or the Pluckthun/Honegger database(http://www.bioc.unizh.ch/antibody/Sequences/Germline s). To compare thehuman sequences to the V regions of VH or VL domains in an antibody ofinterest a sequence alignment algorithm such as available via websiteslike www.expasy.ch/tools/# align can be used, but also manual alignmentwith the limited set of sequences can be performed. Human germline lightand heavy chain sequences of the families with the same combinations ofcanonical folds and with the highest degree of homology with theframework regions 1, 2, and 3 of each chain are selected and comparedwith the variable region of interest; also the FR4 is checked againstthe human germline JH and JK or JL regions. Note that in the calculationof overall percent sequence homology the residues of FR1, FR2 and FR3are evaluated using the closest match sequence from the human germlinefamily with the identical combination of canonical folds. Only residuesdifferent from the closest match or other members of the same familywith the same combination of canonical folds are scored (NB—excludingany primer-encoded differences). However, for the purposes ofhumanization, residues in framework regions identical to members ofother human germline families, which do not have the same combination ofcanonical folds, can be considered “human”, despite the fact that theseare scored “negative” according to the stringent conditions describedabove. This assumption is based on the “mix and match” approach forhumanization, in which each of FR1, FR2, FR3 and FR4 is separatelycompared to its closest matching human germline sequence and thehumanized molecule therefore contains a combination of different FRs aswas done by Qu and colleagues (Qu et al., Clin. Cancer Res. 5:3095-3100(1999)) and Ono and colleagues (Ono et al., Mol. Immunol. 36:387-395(1999)). The boundaries of the individual framework regions may beassigned using the IMGT numbering scheme, which is an adaptation of thenumbering scheme of Chothia (Lefranc et al., NAR 27: 209-212 (1999);http://im.gt.cines.fr). Antibodies with high human homology may comprisehypervariable loops or CDRs having human or human-like canonical folds,as discussed in detail below. In an embodiment at least onehypervariable loop or CDR in either the VH domain or the VL domain ofthe antibody with high human homology may be obtained or derived from aVH or VL domain of a non-human antibody, for example a conventionalantibody from a species of Camelidae, yet exhibit a predicted or actualcanonical fold structure which is substantially identical to a canonicalfold structure which occurs in human antibodies. It is well establishedin the art that although the primary amino acid sequences ofhypervariable loops present in both VH domains and VL domains encoded bythe human germline are, by definition, highly variable, allhypervariable loops, except CDR H3 of the VH domain, adopt only a fewdistinct structural conformations, termed canonical folds (Chothia etal., J. Mol. Biol. 196:901-917 (1987); Tramontano et al. Proteins6:382-94 (1989)), which depend on both the length of the hypervariableloop and presence of the so-called canonical amino acid residues(Chothia et al., J. Mol. Biol. 196:901-917 (1987)). Actual canonicalstructures of the hypervariable loops in intact VH or VL domains can bedetermined by structural analysis (e.g. X-ray crystallography), but itis also possible to predict canonical structure on the basis of keyamino acid residues which are characteristic of a particular structure(discussed further below). In essence, the specific pattern of residuesthat determines each canonical structure forms a “signature” whichenables the canonical structure to be recognised in hypervariable loopsof a VH or VL domain of unknown structure; canonical structures cantherefore be predicted on the basis of primary amino acid sequencealone. The predicted canonical fold structures for the hypervariableloops of any given VH or VL sequence in an antibody with high humanhomology can be analysed using algorithms which are publicly availablefrom www.bioinf.org.uk/abs/chothia.html,www.biochem.ucl.ac.uk/˜martin/antibodies.html andwww.bioc.unizh.ch/antibody/Sequences/Germlines/Vbase_hVk.html. Thesetools permit query VH or VL sequences to be aligned against human VH orVL domain sequences of known canonical structure, and a prediction ofcanonical structure made for the hypervariable loops of the querysequence. In the case of the VH domain, H1 and H2 loops may be scored ashaving a canonical fold structure “substantially identical” to acanonical fold structure known to occur in human antibodies if at leastthe first, and preferable both, of the following criteria are fulfilled:

1. An identical length, determined by the number of residues, to theclosest matching human canonical structural class.

2. At least 33% identity, preferably at least 50% identity with the keyamino acid residues described for the corresponding human H1 and H2canonical structural classes (note for the purposes of the foregoinganalysis the H1 and H2 loops are treated separately and each comparedagainst its closest matching human canonical structural class). Theforegoing analysis relies on prediction of the canonical structure ofthe H1 and H2 loops of the antibody of interest. If the actualstructures of the H1 and H2 loops in the antibody of interest are known,for example based on X-ray crystallography, then the H1 and H2 loops inthe antibody of interest may also be scored as having a canonical foldstructure “substantially identical” to a canonical fold structure knownto occur in human antibodies if the length of the loop differs from thatof the closest matching human canonical structural class (typically by+1 or +2 amino acids) but the actual structure of the H1 and H2 loops inthe antibody of interest matches the structure of a human canonicalfold. Key amino acid residues found in the human canonical structuralclasses for the first and second hypervariable loops of human VH domains(H1 and H2) are described by Chothia et al., J. Mol. Biol. 227:799-817(1992), the contents of which are incorporated herein in their entiretyby reference. In particular, Table 3 on page 802 of Chothia et al.,which is specifically incorporated herein by reference, lists preferredamino acid residues at key sites for H1 canonical structures found inthe human germline, whereas Table 4 on page 803, also specificallyincorporated by reference, lists preferred amino acid residues at keysites for CDR H2 canonical structures found in the human germline. In anembodiment, both H1 and H2 in the VH domain of the antibody with highhuman homology exhibit a predicted or actual canonical fold structurewhich is substantially identical to a canonical fold structure whichoccurs in human antibodies. Antibodies with high human homology maycomprise a VH domain in which the hypervariable loops H1 and H2 form acombination of canonical fold structures which is identical to acombination of canonical structures known to occur in at least one humangermline VH domain. It has been observed that only certain combinationsof canonical fold structures at H1 and H2 actually occur in VH domainsencoded by the human germline. In an embodiment H1 and H2 in the VHdomain of the antibody with high human homology may be obtained from aVH domain of a non-human species, e.g. a Camelidae species, yet form acombination of predicted or actual canonical fold structures which isidentical to a combination of canonical fold structures known to occurin a human germline or somatically mutated VH domain. In non-limitingembodiments H1 and H2 in the VH domain of the antibody with high humanhomology may be obtained from a VH domain of a non-human species, e.g. aCamelidae species, and form one of the following canonical foldcombinations: 1-1, 1-2, 1-3, 1-6, 1-4, 2-1, 3-1 and 3-5. An antibodywith high human homology may contain a VH domain which exhibits bothhigh sequence identity/sequence homology with human VH, and whichcontains hypervariable loops exhibiting structural homology with humanVH. It may be advantageous for the canonical folds present at H1 and H2in the VH domain of the antibody with high human homology, and thecombination thereof, to be “correct” for the human VH germline sequencewhich represents the closest match with the VH domain of the antibodywith high human homology in terms of overall primary amino acid sequenceidentity. By way of example, if the closest sequence match is with ahuman germline VH3 domain, then it may be advantageous for H1 and H2 toform a combination of canonical folds which also occurs naturally in ahuman VH3 domain. This may be particularly important in the case ofantibodies with high human homology which are derived from non-humanspecies, e.g. antibodies containing VH and VL domains which are derivedfrom camelid conventional antibodies, especially antibodies containinghumanised camelid VH and VL domains. Thus, in an embodiment the VHdomain of the GARP antibody with high human homology may exhibit asequence identity or sequence homology of 80% or greater, 85% orgreater, 90% or greater, 95% or greater, 97% or greater, or up to 99% oreven 100% with a human VH domain across the framework regions FR1, FR2,FR3 and FR4, and in addition H1 and H2 in the same antibody are obtainedfrom a non-human VH domain (e.g. derived from a Camelidae species), butform a combination of predicted or actual canonical fold structureswhich is the same as a canonical fold combination known to occurnaturally in the same human VH domain. In other embodiments, L1 and L2in the VL domain of the antibody with high human homology are eachobtained from a VL domain of a non-human species (e.g. a camelid-derivedVL domain), and each exhibits a predicted or actual canonical foldstructure which is substantially identical to a canonical fold structurewhich occurs in human antibodies. As with the VH domains, thehypervariable loops of VL domains of both VLambda and VKappa types canadopt a limited number of conformations or canonical structures,determined in part by length and also by the presence of key amino acidresidues at certain canonical positions. Within an antibody of interesthaving high human homology, L1, L2 and L3 loops obtained from a VLdomain of a non-human species, e.g. a Camelidae species, may be scoredas having a canonical fold structure “substantially identical” to acanonical fold structure known to occur in human antibodies if at leastthe first, and preferable both, of the following criteria are fulfilled:

1. An identical length, determined by the number of residues, to theclosest matching human structural class.

2. At least 33% identity, preferably at least 50% identity with the keyamino acid residues described for the corresponding human L1 or L2canonical structural classes, from either the VLambda or the VKapparepertoire (note for the purposes of the foregoing analysis the L1 andL2 loops are treated separately and each compared against its closestmatching human canonical structural class). The foregoing analysisrelies on prediction of the canonical structure of the L1, L2 and L3loops in the VL domain of the antibody of interest. If the actualstructure of the L1, L2 and L3 loops is known, for example based onX-ray crystallography, then L1, L2 or L3 loops derived from the antibodyof interest may also be scored as having a canonical fold structure“substantially identical” to a canonical fold structure known to occurin human antibodies if the length of the loop differs from that of theclosest matching human canonical structural class (typically by +1 or +2amino acids) but the actual structure of the Camelidae loops matches ahuman canonical fold. Key amino acid residues found in the humancanonical structural classes for the CDRs of human VLambda and VKappadomains are described by Morea et al. Methods, 20: 267-279 (2000) andMartin et al., J. Mol. Biol., 263:800-815 (1996). The structuralrepertoire of the human VKappa domain is also described by Tomlinson etal. EMBO J. 14:4628-4638 (1995), and that of the VLambda domain byWilliams et al. J. Mol. Biol., 264:220-232 (1996). The contents of allthese documents are to be incorporated herein by reference. L1 and L2 inthe VL domain of an antibody with high human homology may form acombination of predicted or actual canonical fold structures which isidentical to a combination of canonical fold structures known to occurin a human germline VL domain. In non-limiting embodiments L1 and L2 inthe VLambda domain of an antibody with high human homology (e.g. anantibody containing a camelid-derived VL domain or a humanised variantthereof) may form one of the following canonical fold combinations:11-7, 13-7(A,B,C), 14-7(A,B), 12-11, 14-11 and 12-12 (as defined inWilliams et al. J. Mol. Biol. 264:220-32 (1996) and as shown onhttp://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVL.html). Innon-limiting embodiments L1 and L2 in the Vkappa domain may form one ofthe following canonical fold combinations: 2-1, 3-1, 4-1 and 6-1 (asdefined in Tomlinson et al. EMBO J. 14:4628-38 (1995) and as shown onhttp://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVK.html).

In a further embodiment, all three of L1, L2 and L3 in the VL domain ofan antibody with high human homology may exhibit a substantially humanstructure. It is preferred that the VL domain of the antibody with highhuman homology exhibit both high sequence identity/sequence homologywith human VL, and also that the hypervariable loops in the VL domainexhibit structural homology with human VL.

In an embodiment, the VL domain of the GARP antibody with high humanhomology may exhibit a sequence identity of 80% or greater, 85% orgreater, 90% or greater, 95% or greater, 97% or greater, or up to 99% oreven 100% with a human VL domain across the framework regions FR1, FR2,FR3 and FR4, and in addition hypervariable loop L1 and hypervariableloop L2 may form a combination of predicted or actual canonical foldstructures which is the same as a canonical fold combination known tooccur naturally in the same human VL domain. It is, of course, envisagedthat VH domains exhibiting high sequence identity/sequence homology withhuman VH, and also structural homology with hypervariable loops of humanVH will be combined with VL domains exhibiting high sequenceidentity/sequence homology with human VL, and also structural homologywith hypervariable loops of human VL to provide antibodies with highhuman homology containing VH/VL pairings (e.g. camelid-derived VH/VLpairings) with maximal sequence and structural homology to human-encodedVH/VL pairings.

“Immunospecific”, “specific for” or to “specifically bind”—As usedherein, an antibody is said to be “immunospecific”, “specific for” or to“specifically bind” an antigen if it reacts at a detectable level withthe antigen, preferably with an affinity constant, Ka, of greater thanor equal to about 10⁴ M⁻¹, or greater than or equal to about 10⁵ M⁻¹,greater than or equal to about 10⁶ M⁻¹, greater than or equal to about10⁷ M⁻¹, or greater than or equal to 10⁸ M⁻¹, or greater than or equalto 10⁹ M⁻¹, or greater than or equal to 10¹⁰ M⁻¹. Affinity of anantibody for its cognate antigen is also commonly expressed as adissociation constant Kd, and in certain embodiments, an antibodyspecifically binds to antigen if it binds with a Kd of less than orequal to 10⁻⁴ M, less than or equal to about 10⁻⁵ M, less than or equalto about 10⁻⁶ M, less than or equal to 10⁻⁷ M, or less than or equal to10⁻⁸ M, or less than or equal to 5.10⁻⁹ M, or less than or equal to 10⁻⁹M, or less than or equal to 5.10⁻¹⁰ M, or less than or equal to 10⁻¹⁰ M.Affinities of antibodies can be readily determined using conventionaltechniques, for example, those described by Scatchard G et al. (Theattractions of proteins for small molecules and ions. Ann NY Acad Sci1949; 51:660-672). Binding properties of an antibody to antigens, cellsor tissues thereof may generally be determined and assessed usingimmunodetection methods including, for example, immunofluorescence-basedassays, such as immuno-histochemistry (IHC) and/orfluorescence-activated cell sorting (FACS).

“Isolated nucleic acid”—As used herein, is a nucleic acid that issubstantially separated from other genome DNA sequences as well asproteins or complexes such as ribosomes and polymerases, which naturallyaccompany a native sequence. The term embraces a nucleic acid sequencethat has been removed from its naturally occurring environment, andincludes recombinant or cloned DNA isolates and chemically synthesizedanalogues or analogues biologically synthesized by heterologous systems.A substantially pure nucleic acid includes isolated forms of the nucleicacid. Of course, this refers to the nucleic acid as originally isolatedand does not exclude genes or sequences later added to the isolatednucleic acid by the hand of man. The term “polypeptide” is used in itsconventional meaning, i.e., as a sequence of amino acids. Thepolypeptides are not limited to a specific length of the product.Peptides, oligopeptides, and proteins are included within the definitionof polypeptide, and such terms may be used interchangeably herein unlessspecifically indicated otherwise. This term also does not refer to orexclude post-expression modifications of the polypeptide, for example,glycosylation, acetylation, phosphorylation and the like, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising CDRs and beingcapable of binding an antigen. An “isolated polypeptide” is one that hasbeen identified and separated and/or recovered from a component of itsnatural environment. In preferred embodiments, the isolated polypeptidewill be purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornon-reducing conditions using Coomassie blue or, preferably, silverstaining. Isolated polypeptide includes the polypeptide in situ withinrecombinant cells since at least one component of the polypeptide'snatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

“Identity” or “identical”—As used herein, the term “identity” or“identical”, when used in a relationship between the sequences of two ormore polypeptides, refers to the degree of sequence relatedness betweenpolypeptides, as determined by the number of matches between strings oftwo or more amino acid residues. “Identity” measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (i.e., “algorithms”). Identity of related polypeptidescan be readily calculated by known methods. Such methods include, butare not limited to, those described in Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987;Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M.Stockton Press, New York, 1991; and Carillo et al., SIAM J. AppliedMath. 48, 1073 (1988). Preferred methods for determining identity aredesigned to give the largest match between the sequences tested. Methodsof determining identity are described in publicly available computerprograms. Preferred computer program methods for determining identitybetween two sequences include the GCG program package, including GAP(Devereux et al., Nucl. Acid. Res. 2, 387 (1984); Genetics ComputerGroup, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, andFASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTXprogram is publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well-knownSmith Waterman algorithm may also be used to determine identity.

“Modified antibody”—As used herein, the term “modified antibody”includes synthetic forms of antibodies which are altered such that theyare not naturally occurring, e.g., antibodies that comprise at least twoheavy chain regions but not two complete heavy chains (such as, domaindeleted antibodies or minibodies); multispecific forms of antibodies(e.g., bispecific, trispecific, etc.) altered to bind to two or moredifferent antigens or to different epitopes on a single antigen); heavychain molecules joined to scFv molecules and the like. ScFv moleculesare known in the art and are described, e.g., in U.S. Pat. No.5,892,019. In addition, the term “modified antibody” includesmultivalent forms of antibodies (e.g., trivalent, tetravalent, etc.,antibodies that bind to three or more copies of the same antigen). Inanother embodiment, a modified antibody of the invention is a fusionprotein comprising at least one heavy chain region lacking a CH2 domainand comprising a binding domain of a polypeptide comprising the bindingregion of one member of a receptor ligand pair.

“Mammal”—As used herein, the term “mammal” refers to any mammal,including humans, domestic and farm animals, and zoo, sports, or petanimals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

“Monoclonal antibody”—As used herein, the term “monoclonal antibody”refers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprised in thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single antigenic site.Furthermore, in contrast to polyclonal antibody preparations thatinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they may be synthesizeduncontaminated by other antibodies. The modifier “monoclonal” is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies useful in the presentinvention may be prepared by the hybridoma methodology first describedby Kohler et al., Nature, 256:495 (1975), or may be made usingrecombinant DNA methods in bacterial, eukaryotic animal or plant cells(see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” mayalso be isolated from phage antibody libraries using the techniquesdescribed in Clackson et al., Nature, 352:624-628 (1991) and Marks etal., J. Mol. Biol., 222:581-597 (1991), for example.

“Native sequence”—As used herein, the term “native sequence” refers to apolynucleotide is one that has the same nucleotide sequence as apolynucleotide derived from nature. A “native sequence” polypeptide isone that has the same amino acid sequence as a polypeptide (e.g.,antibody) derived from nature (e.g., from any species). Such nativesequence polynucleotides and polypeptides can be isolated from nature orcan be produced by recombinant or synthetic means. A polynucleotide“variant”, as the term is used herein, is a polynucleotide thattypically differs from a polynucleotide specifically disclosed herein inone or more substitutions, deletions, additions and/or insertions. Suchvariants may be naturally occurring or may be synthetically generated,for example, by modifying one or more of the polynucleotide sequences ofthe invention and evaluating one or more biological activities of theencoded polypeptide as described herein and/or using any of a number oftechniques well known in the art. A polypeptide “variant”, as the termis used herein, is a polypeptide that typically differs from apolypeptide specifically disclosed herein in one or more substitutions,deletions, additions and/or insertions. Such variants may be naturallyoccurring or may be synthetically generated, for example, by modifyingone or more of the above polypeptide sequences of the invention andevaluating one or more biological activities of the polypeptide asdescribed herein and/or using any of a number of techniques well knownin the art. Modifications may be made in the structure of thepolynucleotides and polypeptides of the present invention and stillobtain a functional molecule that encodes a variant or derivativepolypeptide with desirable characteristics. When it is desired to alterthe amino acid sequence of a polypeptide to create an equivalent, oreven an improved, variant or region of a polypeptide of the invention,one skilled in the art will typically change one or more of the codonsof the encoding DNA sequence. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of its ability to bind other polypeptides (e.g.,antigens) or cells. Since it is the binding capacity and nature of aprotein that defines that protein's biological functional activity,certain amino acid sequence substitutions can be made in a proteinsequence, and of course, its underlying DNA coding sequence, andnevertheless obtain a protein with similar properties. It is thuscontemplated that various changes may be made in the peptide sequencesof the disclosed compositions, or corresponding DNA sequences thatencode said peptides without appreciable loss of their biologicalutility or activity. In many instances, a polypeptide variant willcontain one or more conservative substitutions. A “conservativesubstitution” is one in which an amino acid is substituted for anotheramino acid that has similar properties, such that one skilled in the artof peptide chemistry would expect the secondary structure andhydropathic nature of the polypeptide to be substantially unchanged. Asoutlined above, amino acid substitutions are generally therefore basedon the relative similarity of the amino acid side-chain substituents,for example, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take several of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine. Amino acid substitutions may further be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

“Pharmaceutically acceptable excipient”—As used herein, the term“pharmaceutically acceptable excipient” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. Said excipientdoes not produce an adverse, allergic or other untoward reaction whenadministered to an animal, preferably a human. For human administration,preparations should meet sterility, pyrogenicity, and general safety andpurity standards as required by FDA Office of Biologics standards.

“Specificity”—As used herein, the term “specificity” refers to theability to specifically bind (e.g., immunoreact with) a given target,e.g., GARP. A polypeptide may be monospecific and contain one or morebinding sites which specifically bind a target, or a polypeptide may bemultispecific and contain two or more binding sites which specificallybind the same or different targets. In an embodiment, an antibody of theinvention is specific for more than one target. For example, in anembodiment, a multispecific binding molecule of the invention binds toGARP and a second molecule expressed on a tumor cell. Exemplaryantibodies which comprise antigen binding sites that bind to antigensexpressed on tumor cells are known in the art and one or more CDRs fromsuch antibodies can be included in an antibody of the invention.

“Synthetic”—As used herein the term “synthetic” with respect topolypeptides includes polypeptides which comprise an amino acid sequencethat is not naturally occurring. For example, non-naturally occurringpolypeptides are modified forms of naturally occurring polypeptides(e.g., comprising a mutation such as an addition, substitution ordeletion) or polypeptides which comprise a first amino acid sequence(which may or may not be naturally occurring) that is linked in a linearsequence of amino acids to a second amino acid sequence (which may ormay not be naturally occurring) to which it is not naturally linked innature.

“Single-chain Fv” also abbreviated as “sFv” or “scFv”—As used herein,the terms “Single-chain Fv”, “sFv” or “scFv” are antibody fragments thatcomprise the VH and VL antibody domains connected into a singlepolypeptide chain. Preferably, the sFv polypeptide further comprises apolypeptide linker between the VH and VL domains that enables the sFv toform the desired structure for antigen binding. For a review of sFv, seePluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994);Borrebaeck 1995, infra.

“Variable region” or “variable domain”—As used herein, the term“variable” refers to the fact that certain regions of the variabledomains VH and VL differ extensively in sequence among antibodies andare used in the binding and specificity of each particular antibody forits target antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called “hypervariable loops” in each of the VL domain andthe VH domain which form part of the antigen binding site. The first,second and third hypervariable loops of the VLambda light chain domainare referred to herein as L1 (λ), L2 (λ) and L3 (λ) and may be definedas comprising residues 24-33 (L1(λ), consisting of 9, 10 or 11 aminoacid residues), 49-53 L2 (λ), consisting of 3 residues) and 90-96(L3(λ), consisting of 6 residues) in the VL domain (Morea et al.,Methods 20:267-279 (2000)). The first, second and third hypervariableloops of the VKappa light chain domain are referred to herein as L1(κ),L2(κ) and L3(κ) and may be defined as comprising residues 25-33 (L1(κ),consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2(κ), consistingof 3 residues) and 90-97 (L3(κ), consisting of 6 residues) in the VLdomain (Morea et al., Methods 20:267-279 (2000)). The first, second andthird hypervariable loops of the VH domain are referred to herein as H1,H2 and H3 and may be defined as comprising residues 25-33 (HI,consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4residues) and 91-105 (H3, highly variable in length) in the VH domain(Morea et al., Methods 20:267-279 (2000)). Unless otherwise indicated,the terms L1, L2 and L3 respectively refer to the first, second andthird hypervariable loops of a VL domain, and encompass hypervariableloops obtained from both Vkappa and Vlambda isotypes. The terms H1, H2and H3 respectively refer to the first, second and third hypervariableloops of the VH domain, and encompass hypervariable loops obtained fromany of the known heavy chain isotypes, including [gamma], [epsilon],[delta], a or [mu]. The hypervariable loops L1, L2, L3, H1, H2 and H3may each comprise part of a “complementarity determining region” or“CDR”, as defined below.

“Valency”—As used herein the term “valency” refers to the number ofpotential target binding sites in a polypeptide. Each target bindingsite specifically binds one target molecule or specific site on a targetmolecule. When a polypeptide comprises more than one target bindingsite, each target binding site may specifically bind the same ordifferent molecules (e.g., may bind to different ligands or differentantigens, or different epitopes on the same antigen). The subjectbinding molecules preferably have at least one binding site specific fora human GARP molecule. In particular embodiments the GARP antibodiesprovided herein may be at least bivalent.

“Treating” or “treatment” or “alleviation”—As used herein, the terms“treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures; wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. Those in need of treatment include those already with thedisorder as well as those prone to have the disorder or those in whomthe disorder is to be prevented. A subject or mammal is successfully“treated” for an infection if, after receiving a therapeutic amount ofan antibody according to the methods of the present invention, thepatient shows observable and/or measurable reduction in or absence ofone or more of the following: reduction in the number of pathogeniccells; reduction in the percent of total cells that are pathogenic;and/or relief to some extent, of one or more of the symptoms associatedwith the specific disease or condition; reduced morbidity and mortality,and improvement in quality of life issues. The above parameters forassessing successful treatment and improvement in the disease arereadily measurable by routine procedures familiar to a physician.“TGF-β”—As used herein, the term TGF-β refers to the three isoformsnamed TGF-β1, TGF-β2 and TGF-β3. The peptide structures of the TGF-βisoforms are highly similar (homologies on the order of 70-80%). Theyare all encoded as large protein precursors; TGF-β1 (GenBank Access No:NM_000660 contains 390 amino acids and TGF-β2 (GenBank Access No:NM_001135599 and NM_003238) and TGF-β3 (GenBank Access No: XM_005268028)each contain 412 amino acids. They each have an N-terminal signalpeptide of 20-30 amino acids that they require for secretion from acell, a pro-region (named latency associated peptide or LAP), and a112-114 amino acid C-terminal region that becomes the mature TGF-βmolecule following its release from the pro-region by proteolyticcleavage. After proteolytic cleavage, LAP and mature TGF-β remainnon-covalently associated and form the “latent” TGF-β molecule. In thislatent form, mature TGF-β is prevented from binding to the TGF-βreceptor by LAP. To exert a signal, mature TGF-β must be released fromLAP. Mature TGF-β that is not associated to LAP is called active TGF-β,as it can bind to the TGF-β receptor and transduce a signal.

TGF-β1 has the following amino acid sequence:

(SEQ ID NO: 53) MPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS.

LAP has the following amino acid sequence:

(SEQ ID NO: 54) LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRR.

Mature TGF-β1 has the following amino acid sequence:

(SEQ ID NO: 55) ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQ LSNMIVRSCKCS.

One object of the invention is a protein binding to GARP in the presenceof TGF-β. Another object of the invention is a protein comprising anantigen binding domain, wherein the antigen binding domain bindsspecifically to GARP in the presence of TGF-β.

In an embodiment, said protein binds to GARP only in the presence ofTGF-β.

GARP is also called Leucin Rich Repeat Containing 32 (LRRC32) andbelongs to the Leucin Rich Repeat family. The complete amino acidsequence of the human GARP protein transcript variant 2 of the presentinvention (SEQ ID NO: 1) (GenBank Accession NM_001128922) is:

MRPQILLLLALLTLGLAAQHQDKVPCKMVDKKVSCQVLGLLQVPSVLPPDTETLDLSGNQLRSILASPLGFYTALRHLDLSTNEISFLQPGAFQALTHLEHLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLSLAENSLTRLTRHTFRDMPALEQLDLHSNVLMDIEDGAFEGLPRLTHLNLSRNSLTCISDFSLQQLRVLDLSCNSIEAFQTASQPQAEFQLTWLDLRENKLLHFPDLAALPRLIYLNLSNNLIRLPTGPPQDSKGIHAPSEGWSALPLSAPSGNASGRPLSQLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRNCLRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLRTLLLQGNALRDLPPYTFANLASLQRLNLQGNRVSPCGGPDEPGPSGCVAFSGITSLRSLSLVDNEIELLRAGAFLHTPLTELDLSSNPGLEVATGALGGLEASLEVLALQGNGLMVLQVDLPCFICLKRLNLAENRLSHLPAWTQAVSLEVLDLRNNSFSLLPGSAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFSSQEEVSLSHVRPEDCEKGGLKNINLIIILTFILVSAILLTTLAACCC VRRQKFNQQYKA.

In an embodiment, the protein of the invention binds to GARP when GARPis complexed to TGF-β.

In another embodiment, the protein of the invention binds to GARP whenGARP is complexed to latent TGF-β.

In another embodiment, the protein of the invention binds to a complexof GARP and TGF-β.

In an embodiment, the protein of the invention binds to a complex ofGARP and TGF-β1; TGF-β2, isoform 1; TGF-β2, isoform 2; TGF-β.Preferably, the protein of the invention binds to a complex of GARP andTGF-β1.

In another embodiment, the protein of the invention binds to a complexof GARP and latent TGF-β.

The term “latent TGF-β” as used herein comprises a complex whoseC-terminal fragment, or mature TGF-β1, remains non-covalently bound tothe N-terminal fragment known as LAP.

In another embodiment, the protein of the invention binds to a complexof GARP and latent TGF-β at a KD (the equilibrium dissociation constantbetween the antibody and its antigen) of less than 10⁻¹⁰ M.

In an embodiment, said protein is an antibody molecule selected from thegroup consisting of a whole antibody, a humanized antibody, a singlechain antibody, a dimeric single chain antibody, a Fv, a Fab, a F(ab)′2,a defucosylated antibody, a bi-specific antibody, a diabody, a triabody,a tetrabody.

In another embodiment, said protein is an antibody fragment selectedfrom the group consisting of a unibody, a domain antibody, and ananobody.

In another embodiment, said protein is an antibody mimetic selected fromthe group consisting of an affibody, an affilin, an affitin, anadnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, afynomer, a versabody and a duocalin.

A domain antibody is well known in the art and refers to the smallestfunctional binding units of antibodies, corresponding to the variableregions of either the heavy or light chains of antibodies.

A nanobody is well known in the art and refers to an antibody-derivedtherapeutic protein that contains the unique structural and functionalproperties of naturally-occurring heavy chain antibodies. These heavychain antibodies contain a single variable domain (VHH) and two constantdomains (CH2 and CH3).

A unibody is well known in the art and refers to an antibody fragmentlacking the hinge region of IgG4 antibodies. The deletion of the hingeregion results in a molecule that is essentially half the size oftraditional IgG4 antibodies and has a univalent binding region ratherthan the bivalent biding region of IgG4 antibodies.

An affibody is well known in the art and refers to affinity proteinsbased on a 58 amino acid residue protein domain, derived from one of theIgG binding domain of staphylococcal protein A.

DARPins (Designed Ankyrin Repeat Proteins) are well known in the art andrefer to an antibody mimetic DRP (designed repeat protein) technologydeveloped to exploit the binding abilities of non-antibody polypeptides.

Anticalins are well known in the art and refer to another antibodymimetic technology, wherein the binding specificity is derived fromlipocalins. Anticalins may also be formatted as dual targeting protein,called Duocalins.

Avimers are well known in the art and refer to another antibody mimetictechnology.

Versabodies are well known in the art and refer to another antibodymimetic technology. They are small proteins of 3-5 kDa with >15%cysteines, which form a high disulfide density scaffold, replacing thehydrophobic core the typical proteins have.

In another embodiment, said protein is an immunoconjugate comprising anantibody or fragment thereof conjugated to a therapeutic agent.

In another embodiment, said protein is a conjugate comprising theprotein of the invention conjugated to an imaging agent. Said proteincould be used for example for imaging applications.

Another object of the invention is a protein that binds to GARP andinhibits TGF-β signaling.

In an embodiment, said protein binds to GARP when GARP is complexed toTGF-β.

In another embodiment, said protein binds to GARP when GARP is complexedto latent TGF-β.

In another embodiment, said protein binds to a complex of GARP andTGF-β.

In another embodiment, said protein binds to a complex of GARP andlatent TGF-β.

In an embodiment, said protein is an antibody molecule selected from thegroup consisting of a whole antibody, a humanized antibody, a singlechain antibody, a dimeric single chain antibody, a Fv, a Fab, a F(ab)′2,a defucosylated antibody, a bi-specific antibody, a diabody, a triabody,a tetrabody.

In another embodiment, said protein is an antibody fragment selectedfrom the group consisting of a unibody, a domain antibody, and ananobody.

In another embodiment, said protein is an antibody mimetic selected fromthe group consisting of an affibody, an affilin, an affitin, anadnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, afynomer, a versabody and a duocalin.

In an embodiment, said protein is an anti-hGARP (anti human GARP)antibody or antigen binding fragment thereof that inhibits TGF-βsignaling.

In an embodiment, said protein prevents or inhibits active TGF-β to bereleased or inhibits the release of mature TGF-β from GARP/TGF-β.

In an embodiment, said protein prevents or inhibits the release ofactive TGF-β from membrane-bound GARP/TGF-β.

In an embodiment, said protein prevents or inhibits active TGF-β to bereleased or inhibits the release of mature TGF-β from Tregs.

In another embodiment, said protein inhibits or prevents mature TGF-β tobind to TGF-β receptors.

In another embodiment, said protein inhibits TGF-β activity and/or theactivation of molecules from the TGF-β receptor signaling pathway.

As used herein, the term “inhibit” means that the protein is capable ofblocking, reducing, preventing or neutralizing TGF-β signaling or therelease of mature TGF-β from Tregs or the binding of mature TGF-β toTGF-β receptors or TGF-β activity and/or the activation of moleculesfrom the TGF-β receptor signaling pathway.

In an embodiment, said protein is a monoclonal antibody.

In another embodiment, said protein is a polyclonal antibody.

In an embodiment, said protein binds to a conformational epitope.

In an embodiment, said conformational epitope comprises one or moreamino acids of hGARP.

In another embodiment, said conformational epitope comprises an epitopeof GARP modified as a result of GARP being complexed with latent TGF-β.In another embodiment, said conformational epitope comprises amino acidsof hGARP and amino acids of latent TGF-β.

In another embodiment, said conformational epitope is a mixedconformational epitope and comprises amino acids from both GARP andTGF-β.

In another embodiment, said conformational epitope is a binding-inducedconformational epitope and comprises amino acids from GARP only, butthat adopts a different conformation in the presence of TGF-β.

In an embodiment, said epitope comprises one or more residues from 101to 141 residues of hGARP amino acid sequence (SEQ ID NO: 1).

These 101 to 141 residues are as set forth in SEQ ID NO: 12:HLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSGLL.

In another embodiment of the invention, said epitope comprises theresidues 137, 138 and 139: YSG of hGARP amino acid sequence (SEQ ID NO:1).

In another embodiment of the invention, said epitope comprises theresidues 137, 138 and 139: YSG of hGARP amino acid sequence (SEQ IDNO: 1) and requires the presence of TGF-β.

In another embodiment of the invention, said epitope comprises theresidues 137, 138 and 139: YSG of hGARP amino acid sequence (SEQ IDNO: 1) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 contiguous residues in N-terminal and/or C-terminal of theresidues 137, 138 and 139: YSG of SEQ ID NO: 1.

In another embodiment of the invention, said epitope comprises theresidues 137, 138 and 139: YSG of hGARP amino acid sequence (SEQ IDNO: 1) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 contiguous residues in N-terminal and/or C-terminal of theresidues 137, 138 and 139: YSG of SEQ ID NO: 1, and requires thepresence of TGF-β.

In an embodiment of the invention, the protein of the invention binds toepitopes preferably within the region 101-141 of hGARP and inhibits therelease of latent TGF-β from GARP.

One skilled in the art can determine the ability of a protein to inhibitTGF-β signaling by measuring for example activation of molecules fromthe TGF-β receptor signaling pathway. One example of such test is themeasurement of the phosphorylation of SMAD2 (as shown in Example 2 ofthe present invention).

Another object of the invention is a protein binding to an epitope of acomplex formed by human GARP and TGF-β, said epitope comprising at leastone of the residues 137, 138, or 139 of GARP (SEQ ID NO: 1) and at leastone residue of TGF-β (SEQ ID NO: 53).

In one embodiment, said protein is an antibody or an antigen bindingfragment thereof.

In another embodiment, said antibody or antigen binding fragment thereofis selected from the group consisting of a whole antibody, a humanizedantibody, a single chain antibody, a dimeric single chain antibody, aFv, a Fab, a F(ab)′2, a defucosylated antibody, a bi-specific antibody,a diabody, a triabody, a tetrabody; or an antibody fragment selectedfrom the group consisting of a unibody, a domain antibody, and ananobody; or an antibody mimetic selected from the group consisting ofan affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin,a DARPin, an anticalin, an avimer, a fynomer, a versabody and aduocalin.

In one embodiment, the epitope comprises one, two or three of theresidues 137, 138, and 139 of GARP (SEQ ID NO: 1).

In another embodiment, the epitope comprises at least one of theresidues 137, 138, or 139 of GARP (SEQ ID NO: 1) and at least oneresidue from the Latency associated peptide (LAP) of TGF-β (SEQ ID NO:54) and at least one residue from mature TGF-β (SEQ ID NO: 55).

In another embodiment, the epitope comprises at least one of theresidues 137, 138, or 139 of GARP (SEQ ID NO: 1) and at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20residue(s) from the Latency associated peptide (LAP) (SEQ ID NO: 54) andat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 residue(s) from mature TGF-β (SEQ ID NO: 55).

In another embodiment, the epitope comprises one, two or three of theresidues 137, 138, and 139 of GARP (SEQ ID NO: 1) and at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20residue(s) from the Latency associated peptide (LAP) (SEQ ID NO: 54) andat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 residue(s) from mature TGF-β (SEQ ID NO: 55).

In another embodiment, the epitope comprises one, two or three of theresidues 137, 138, and 139 of GARP (SEQ ID NO: 1) and at least 1, 2, 3,4, 5, 6, 7, or 8 residue(s) from the Latency associated peptide (LAP)(SEQ ID NO: 54) selected from the group of residues 58, 100, 146, 269,270, 271, 272, 273 of TGF-β (SEQ ID NO: 53) and at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residue(s)from mature TGF-β (SEQ ID NO: 55).

In another embodiment, the epitope comprises one, two or three of theresidues 137, 138, and 139 of GARP (SEQ ID NO: 1) and at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20residue(s) from the Latency associated peptide (LAP) (SEQ ID NO: 54) andat least 1, 2, 3, 4, 5, or 6 residue(s) from mature TGF-β (SEQ ID NO:55) selected from the group of residues 284, 336, 337, 338, 341, and 345of TGF β (SEQ ID NO: 53).

In another embodiment, the epitope comprises one, two or three of theresidues 137, 138, and 139 of GARP (SEQ ID NO: 1) and at least 1, 2, 3,4, 5, 6, 7, or 8 residue(s) from the Latency associated peptide (LAP)(SEQ ID NO: 54) selected from the group of residues 58, 100, 146, 269,270, 271, 272, and 273 of TGF-β (SEQ ID NO: 53) and at least 1, 2, 3, 4,5, or 6 residue(s) from mature TGF-β (SEQ ID NO: 55) selected from thegroup of residues 284, 336, 337, 338, 341, and 345 of TGF β (SEQ ID NO:53).

In another embodiment, the epitope comprises one, two or three of theresidues 137, 138, and 139 of GARP (SEQ ID NO: 1) and at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 residue(s) selected from thegroup of residues 58, 100, 146, 269, 270, 271, 272, 273, 284, 336, 337,338, 341, and 345 of TGF-β (SEQ ID NO: 53).

In another embodiment, the epitope comprises at least one, two or threeof the residues 137, 138 and 139 of GARP (SEQ ID NO: 1) and at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19residue(s) selected from the group of residues 113, 114, 116, 117, 118,119, 140, 142, 143, 144, 145, 146, 162, 163, 165, 166, 167, 170 and 189of GARP (SEQ ID NO: 1) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 residue(s) from the Latencyassociated peptide (LAP) (SEQ ID NO: 54) and at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residue(s) frommature TGF-β (SEQ ID NO: 55).

In another embodiment, the epitope comprises at least one, two or threeof the residues 137, 138 and 139 of GARP (SEQ ID NO: 1) and at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19residue(s) selected from the group of residues 113, 114, 116, 117, 118,119, 140, 142, 143, 144, 145, 146, 162, 163, 165, 166, 167, 170 and 189of GARP (SEQ ID NO: 1) and at least 1, 2, 3, 4, 5, 6, 7, or 8 residue(s)from the Latency associated peptide (LAP) selected from the group ofresidues 58, 100, 146, 269, 270, 271, 272, and 273 of TGF-β and at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20residue(s) from mature TGF-β (SEQ ID NO: 55).

In another embodiment, the epitope comprises at least one, two or threeof the residues 137, 138 and 139 of GARP (SEQ ID NO: 1) and at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19residue(s) selected from the group of residues 113, 114, 116, 117, 118,119, 140, 142, 143, 144, 145, 146, 162, 163, 165, 166, 167, 170 and 189of GARP (SEQ ID NO: 1) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 residue(s) from the Latencyassociated peptide (LAP) (SEQ ID NO: 54) and at least 1, 2, 3, 4, 5, or6 residue(s) from mature TGF-β (SEQ ID NO: 55) selected from the groupof residues 284, 336, 337, 338, 341, and 345 of TGF β (SEQ ID NO: 53).

In another embodiment, the epitope comprises at least one, two or threeof the residues 137, 138 and 139 of GARP (SEQ ID NO: 1) and at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19residue(s) selected from the group of residues 113, 114, 116, 117, 118,119, 140, 142, 143, 144, 145, 146, 162, 163, 165, 166, 167, 170 and 189of GARP (SEQ ID NO: 1) and at least 1, 2, 3, 4, 5, 6, 7, or 8 residue(s)from the Latency associated peptide (LAP) selected from the group ofresidues 58, 100, 146, 269, 270, 271, 272, and 273 of TGF-β and at least1, 2, 3, 4, 5, and 6 residue(s) from mature TGF-β (SEQ ID NO: 55)selected from the group of residues 284, 336, 337, 338, 341, and 345 ofTGF β (SEQ ID NO: 53).

An object of the invention is an antibody against human GARP or antigenbinding fragment thereof wherein the variable region of the heavy chaincomprises at least one of the followings CDRs:

(SEQ ID NO: 2) VH-CDR1: GFSLTGYGIN or (SEQ ID NO: 52) GYGIN; (SEQ ID NO:3) VH-CDR2: MIWSDGSTDYNSVLTS; and (SEQ ID NO: 4) VH-CDR3: DRNYYDYDGAMDY.

Another object of the invention is an anti-hGARP antibody or antigenbinding fragment thereof wherein the variable region of the light chaincomprises at least one of the followings CDRs:

(SEQ ID NO: 5) VL-CDR1: KASDHIKNWLA; (SEQ ID NO: 6) VL-CDR2: GATSLEA;and (SEQ ID NO: 7) VL-CDR3: QQYWSTPWT.

Another object of the invention is an antibody against human GARP orantigen binding fragment thereof wherein the variable region of theheavy chain comprises at least one of the followings CDRs:

(SEQ ID NO: 13) VH-CDR1: SYYID; (SEQ ID NO: 14) VH-CDR2:RIDPEDGGTKYAQKFQG; and (SEQ ID NO: 15) VH-CDR3: NEWETVVVGDLMYEYEY.

Another object of the invention is an anti-hGARP antibody or antigenbinding fragment thereof wherein the variable region of the light chaincomprises at least one of the followings CDRs:

-   -   VL-CDR1: QASQX₁I X₂S X₃LA (SEQ ID NO: 16), wherein X₁ is S or T,        X₂ is S or V, X₃ is Y or F;    -   VL-CDR2: X₁X₂SX₃X₄X₅T (SEQ ID NO: 17), wherein X₁ is G or R; X₂        is A or T; X₃ is R or I; X₄ is L or P; X₅ is Q or K; and    -   VL-CDR3: QQYX₁SX₂PX₃T, wherein X₁ is D, A, Y or V; X₂ is A, L or        V; X₃ is V or P (SEQ ID NO: 18).

Another object of the invention is an anti-hGARP antibody or antigenbinding fragment thereof wherein the variable region of the heavy chaincomprises the VH-CDR1 of SEQ ID NO: 13, VH-CDR2 of SEQ ID NO: 14 andVH-CDR3 of SEQ ID NO: 15 and the variable region of the light chaincomprises at least one of VL-CDR1 as set forth in SEQ ID NO: 19; SEQ IDNO: 22; SEQ ID NO: 25; SEQ ID NO: 28; or SEQ ID NO: 31; at least one ofVL-CDR2 as set forth in SEQ ID NO: 20; SEQ ID NO: 23; SEQ ID NO: 26; SEQID NO: 29; or SEQ ID NO: 32 and at least one of VL-CDR3 as set forth inSEQ ID NO: 21; SEQ ID NO: 24; SEQ ID NO: 27; SEQ ID NO: 30; or SEQ IDNO: 33.

Another object of the invention is an anti-hGARP antibody or antigenbinding fragment thereof wherein the variable region of the light chaincomprises at least one of the followings CDRs:

(SEQ ID NO: 19) VL-CDR1: QASQSISSYLA; (SEQ ID NO: 20) VL-CDR2: GASRLQT;and (SEQ ID NO: 21) VL-CDR3: QQYDSLPVT.

Another object of the invention is an anti-hGARP antibody or antigenbinding fragment thereof wherein the variable region of the light chaincomprises at least one of the followings CDRs:

(SEQ ID NO: 22) VL-CDR1: QASQSIVSYLA; (SEQ ID NO: 23) VL-CDR2: GASRLQT;and (SEQ ID NO: 24) VL-CDR3: QQYASAPVT.

Another object of the invention is an anti-hGARP antibody or antigenbinding fragment thereof wherein the variable region of the light chaincomprises at least one of the followings CDRs:

(SEQ ID NO: 25) VL-CDR1: QASQSISSYLA; (SEQ ID NO: 26) VL-CDR2: GTSRLKT;and (SEQ ID NO: 27) VL-CDR3: QQYYSAPVT.

Another object of the invention is an anti-hGARP antibody or antigenbinding fragment thereof wherein the variable region of the light chaincomprises at least one of the followings CDRs:

VL-CDR1: (SEQ ID NO: 28) QASQTISSFLA; VL-CDR2: (SEQ ID NO: 29) RASIPQT;and VL-CDR3: (SEQ ID NO: 30) QQYVSAPPT.

Another object of the invention is an anti-hGARP antibody or antigenbinding fragment thereof wherein the variable region of the light chaincomprises at least one of the followings CDRs:

VL-CDR1: (SEQ ID NO: 31) QASQSISSYLA; VL-CDR2: (SEQ ID NO: 32) GASRLKT;and VL-CDR3: (SEQ ID NO: 33) QQYASVPVT.

In an embodiment of the invention, the anti-hGARP antibody or antigenbinding fragment thereof may comprise the CH1 domain, hinge region, CH2domain and CH3 domain of a human antibody, in particular IgG1, IgG2,IgG3 or IgG4.

In an embodiment of the invention, the anti-hGARP antibody or antigenbinding fragment thereof comprises in its heavy chain the followingCDRs: VH-CDR1 GFSLTGYGIN (SEQ ID NO: 2), VH-CDR2 MIWSDGSTDYNSVLTS (SEQID NO: 3) and VH-CDR3 DRNYYDYDGAMDY (SEQ ID NO: 4).

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises in its heavy chain thefollowing CDRs: VH-CDR1 GYGIN (SEQ ID NO: 52), VH-CDR2 MIWSDGSTDYNSVLTS(SEQ ID NO: 3) and VH-CDR3 DRNYYDYDGAMDY (SEQ ID NO: 4).

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises in its light chain thefollowing CDRs: VL-CDR1 KASDHIKNWLA (SEQ ID NO: 5), VL-CDR2 GATSLEA (SEQID NO: 6) and VL-CDR3 QQYWSTPWT (SEQ ID NO: 7).

In an embodiment of the invention, the anti-hGARP antibody or antigenbinding fragment thereof comprises in its heavy chain the followingCDRs: VH-CDR1 SYYID (SEQ ID NO: 13), VH-CDR2 RIDPEDGGTKYAQKFQG (SEQ IDNO: 14) and VH-CDR3 NEWETVVVGDLMYEYEY (SEQ ID NO: 15).

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises in its light chain thefollowing CDRs: VL-CDR1 QASQX₁I X₂SX₃LA (SEQ ID NO: 16), wherein X₁ is Sor T, X₂ is S or V, X₃ is Y or F; VL-CDR2 X₁X₂SX₃X₄X₅T (SEQ ID NO: 17),wherein X₁ is G or R; X₂ is A or T; X₃ is R or I; X₄ is L or P; X₅ is Qor K; and VL-CDR3 QQYX₁SX₂PX₃T, wherein X₁ is D, A, Y or V; X₂ is A, Lor V; X₃ is V or P (SEQ ID NO: 18).

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises in its light chain thefollowing CDRs: VL-CDR1 QASQSISSYLA (SEQ ID NO: 19), VL-CDR2 GASRLQT(SEQ ID NO: 20), and VL-CDR3 QQYDSLPVT (SEQ ID NO: 21).

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises in its light chain thefollowing CDRs: VL-CDR1 QASQSIVSYLA (SEQ ID NO: 22); VL-CDR2 GASRLQT(SEQ ID NO: 23); and VL-CDR3: QQYASAPVT (SEQ ID NO: 24).

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises in its light chain thefollowing CDRs: VL-CDR1 QASQSISSYLA (SEQ ID NO: 25); VL-CDR2 GTSRLKT(SEQ ID NO: 26); and VL-CDR3 QQYYSAPVT (SEQ ID NO: 27).

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises in its light chain thefollowing CDRs: VL-CDR1 QASQTISSFLA (SEQ ID NO: 28); VL-CDR2 RASIPQT(SEQ ID NO: 29); and VL-CDR3 QQYVSAPPT (SEQ ID NO: 30).

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises in its light chain thefollowing CDRs: VL-CDR1 QASQSISSYLA (SEQ ID NO: 31); VL-CDR2 GASRLKT(SEQ ID NO: 32); and VL-CDR3 QQYASVPVT (SEQ ID NO: 33).

According to the invention, any of the CDRs 1, 2 and 3 of the heavy andlight chains may be characterized as having an amino acid sequence thatshares at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity with the particular CDR or sets of CDRs listed in thecorresponding SEQ ID NO.

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof is selected from the group consistingof an antibody having:

-   -   (i) the heavy chain CDR 1, 2 and 3 (VH-CDR1, VH-CDR2, VH-CDR3)        amino acid sequences as shown in SEQ ID NO: 2, 3 and 4; and    -   (ii) the light chain CDR 1, 2 and 3 (VL-CDR1, VL-CDR2, VL-CDR3)        amino acid sequences as shown in SEQ ID NO: 5, 6 and 7        respectively;

optionally wherein one, two, three or more of the amino acids in any ofsaid sequences may be substituted by a different amino acid.

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof is selected from the group consistingof an antibody having:

-   -   (i) the heavy chain CDR 1, 2 and 3 (VH-CDR1, VH-CDR2, VH-CDR3)        amino acid sequences as shown in SEQ ID NO: 52, 3 and 4; and    -   (ii) the light chain CDR 1, 2 and 3 (VL-CDR1, VL-CDR2, VL-CDR3)        amino acid sequences as shown in SEQ ID NO: 5, 6 and 7        respectively;

optionally wherein one, two, three or more of the amino acids in any ofsaid sequences may be substituted by a different amino acid.

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof is selected from the group consistingof an antibody having:

-   -   (i) the heavy chain CDR 1, 2 and 3 (VH-CDR1, VH-CDR2, VH-CDR3)        amino acid sequences as shown in SEQ ID NO: 13, 14 and 15; and    -   (ii) the light chain CDR 1, 2 and 3 (VL-CDR1, VL-CDR2, VL-CDR3)        amino acid sequences as shown in SEQ ID NO: 16, 17 and 18        respectively;

optionally wherein one, two, three or more of the amino acids in any ofsaid sequences may be substituted by a different amino acid.

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises:

-   -   (i) the heavy chain CDR 1, 2 and 3 (VH-CDR1, VH-CDR2, VH-CDR3)        amino acid sequences as shown in SEQ ID NO: 13, 14 and 15; and    -   (ii) the light chain CDR 1, 2 and 3 (VL-CDR1, VL-CDR2, VL-CDR3)        amino acid sequences as shown in SEQ ID NO: 19, 20 and 21        respectively;

optionally wherein one, two, three or more of the amino acids in any ofsaid sequences may be substituted by a different amino acid.

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises:

-   -   (i) the heavy chain CDR 1, 2 and 3 (VH-CDR1, VH-CDR2, VH-CDR3)        amino acid sequences as shown in SEQ ID NO: 13, 14 and 15; and    -   (ii) the light chain CDR 1, 2 and 3 (VL-CDR1, VL-CDR2, VL-CDR3)        amino acid sequences as shown in SEQ ID NO: 22, 23 and 24        respectively;

optionally wherein one, two, three or more of the amino acids in any ofsaid sequences may be substituted by a different amino acid.

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises:

-   -   (i) the heavy chain CDR 1, 2 and 3 (VH-CDR1, VH-CDR2, VH-CDR3)        amino acid sequences as shown in SEQ ID NO: 13, 14 and 15; and    -   (ii) the light chain CDR 1, 2 and 3 (VL-CDR1, VL-CDR2, VL-CDR3)        amino acid sequences as shown in SEQ ID NO: 25, 26 and 27        respectively;

optionally wherein one, two, three or more of the amino acids in any ofsaid sequences may be substituted by a different amino acid.

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises:

-   -   (i) the heavy chain CDR 1, 2 and 3 (VH-CDR1, VH-CDR2, VH-CDR3)        amino acid sequences as shown in SEQ ID NO: 13, 14 and 15; and    -   (ii) the light chain CDR 1, 2 and 3 (VL-CDR1, VL-CDR2, VL-CDR3)        amino acid sequences as shown in SEQ ID NO: 28, 29 and 30        respectively;

optionally wherein one, two, three or more of the amino acids in any ofsaid sequences may be substituted by a different amino acid.

In another embodiment of the invention, the anti-hGARP antibody orantigen binding fragment thereof comprises:

-   -   (i) the heavy chain CDR 1, 2 and 3 (VH-CDR1, VH-CDR2, VH-CDR3)        amino acid sequences as shown in SEQ ID NO: 13, 14 and 15; and    -   (ii) the light chain CDR 1, 2 and 3 (VL-CDR1, VL-CDR2, VL-CDR3)        amino acid sequences as shown in SEQ ID NO: 31, 32 and 33        respectively;

optionally wherein one, two, three or more of the amino acids in any ofsaid sequences may be substituted by a different amino acid.

In an embodiment, the anti-hGARP antibody or antigen binding fragmentthereof comprises a variable heavy chain CDR3 comprising an amino acidsequence of SEQ ID NO: 4 (DRNYYDYDGAMDY), or sequence variant thereof,wherein the sequence variant comprises one, two or three amino acidsubstitutions in the recited sequence.

In an embodiment, the anti-hGARP antibody or antigen binding fragmentthereof comprises a variable heavy chain CDR3 comprising an amino acidsequence of SEQ ID NO: 15, or sequence variant thereof, wherein thesequence variant comprises one, two or three amino acid substitutions inthe recited sequence.

Another object of the invention is the anti-hGARP antibody MHGARP8 orantigen binding fragment thereof comprising a heavy chain variableregion of sequence SEQ ID NO: 8 and a light chain variable region ofsequence SEQ ID NO: 9.

(SEQ ID NO: 8) MAVLALLFCLVTFPSCILSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGINWVRQPPGKGLEWLGMIWSDGSTDYNSVLTSRLRISKDNSNSQVFLKMNSLQVDDTARYYCARDRNYYDYDGAMDYWGQGTSVTVSS. (SEQ ID NO: 9)MKFPSQLLLFLLFRITGIICDIQVTQSSSYLSVSLGDRVTITCKASDHIKNWLAWYQQKPGIAPRLLVSGATSLEAGVPSRFSGSGSGKNFTLSITSLQTEDVATYYCQQYWSTPWTFGGGTTLEIR.

Another object of the invention is the anti-hGARP antibody MHGARP8 orantigen binding fragment thereof comprising a heavy chain variableregion of sequence SEQ ID NO: 50 and a light chain variable region ofsequence SEQ ID NO: 51, wherein SEQ ID NO: 50 and SEQ ID NO: 51correspond, respectively, to SEQ ID NO: 8 and SEQ ID NO: 9 wherein thesignal peptide sequences were removed.

(SEQ ID NO: 50) QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGINWVRQPPGKGLEWLGMIWSDGSTDYNSVLTSRLRISKDNSNSQVFLKMNSLQVDDTARYYCARDRNYYDYDGAMDYWGQGTSVTVSS. (SEQ ID NO: 51)DIQVTQSSSYLSVSLGDRVTITCKASDHIKNWLAWYQQKPGIAPRLLVSGATSLEAGVPSRFSGSGSGKNFTLSITSLQTEDVATYYCQQYWSTPWTFGG GTTLEIR.

Another object of the invention is the anti-hGARP antibody LHG10 orantigen binding fragment thereof comprising a heavy chain variableregion of sequence SEQ ID NO: 34 and a light chain variable region ofsequence SEQ ID NO: 35.

(SEQ ID NO: 34) EVQLVQPGAELRNSGASVKVSCKASGYRFTSYYIDWVRQAPGQGLEWMGRIDPEDGGTKYAQKFQGRVTFTADTSTSTAYVELSSLRSEDTAVYYCARNEWETVVVGDLMYEYEYWGQGTQVTVSS. (SEQ ID NO: 35)DIQMTQSPTSLSASLGDRVTITCQASQSISSYLAWYQQKPGQAPKLLIYGASRLQTGVPSRFSGSGSGTSFTLTISGLEAEDAGTYYCQQYDSLPVTFGQ GTKVELK.

Another object of the invention is the anti-hGARP antibody LHG10.3 orantigen binding fragment thereof comprising a heavy chain variableregion of sequence SEQ ID NO: 34 and a light chain variable region ofsequence SEQ ID NO: 36.

(SEQ ID NO: 36) DIQMTQSPSSLSASLGDRVTITCQASQSIVSYLAWYQQKPGQAPKLLIYGASRLQTGVPSRFSGSGSGTSFTLTISGLEAEDAGTYYCQQYASAPVTFGQ GTGVELK.

Another object of the invention is the anti-hGARP antibody LHG10.4 orantigen binding fragment thereof comprising a heavy chain variableregion of sequence SEQ ID NO: 34 and a light chain variable region ofsequence SEQ ID NO: 37.

(SEQ ID NO: 37) DIQMTQSPSSLSASLGDRVTITCQASQSISSYLAWYQQKPGQAPKLLIYGTSRLKTGVPSRFSGSGSGTSFTLTISGLEAEDAGTYYCQQYYSAPVTFGQ GTKVELK.

Another object of the invention is the anti-hGARP antibody LHG10.5 orantigen binding fragment thereof comprising a heavy chain variableregion of sequence SEQ ID NO: 34 and a light chain variable region ofsequence SEQ ID NO: 38.

(SEQ ID NO: 38) DIQMTQSPSSLSPSLGDRVTITCQASQTISSFLAWYHQKPGQPPKLLIYRASIPQTGVPSRFSGSGSGTSFTLTIGGLEAEDAGTYYCQQYVSAPPTFGQ GTKVELK.

Another object of the invention is the anti-hGARP antibody LHG10.6thereof comprising a heavy chain variable region of sequence SEQ ID NO:34 and a light chain variable region of sequence SEQ ID NO: 39.

(SEQ ID NO: 39) DIQMTQSPSSLSASLGDRVTITCQASQSISSYLAWYQQKPGQAPNILIYGASRLKTGVPSRFSGSGSGTSFTLTISGLEAEDAGTYYCQQYASVPVTFGQ GTKVELK.

In an embodiment of the invention, one, two, three or more of the aminoacids of the heavy chain or light chain variable regions as describedhere above may be substituted by a different amino acid.

In another embodiment, an antibody of the invention comprises heavy andlight chain variable regions comprising amino acid sequences that arehomologous to the amino acid sequences of the MHGARP8 antibody describedherein, wherein the antibodies retain the desired functional propertiesof the protein of the invention.

In an embodiment of the invention, the sequence of the heavy chainvariable region of an anti-hGARP antibody of the invention encompassessequences that have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% identity with SEQ ID NO: 8 or with SEQ ID NO: 50.

In an embodiment of the invention, the sequence of light chain variableregion of an anti-hGARP antibody of the invention encompasses sequencesthat have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identitywith SEQ ID NO: 9 or with SEQ ID NO: 51.

In another embodiment, an antibody of the invention comprises heavy andlight chain variable regions comprising amino acid sequences that arehomologous to the amino acid sequences of the LHG10 antibody describedherein, and wherein the antibodies retain the desired functionalproperties of the protein of the invention.

In an embodiment of the invention, the sequence of the heavy chainvariable region of an anti-hGARP antibody of the invention encompassessequences that have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% identity with SEQ ID NO: 34.

In an embodiment of the invention, the sequence of light chain variableregion of an anti-hGARP antibody of the invention encompasses sequencesthat have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identitywith SEQ ID NO: 35; 36; 37; 38 or 39.

In any of the antibodies of the invention, e.g. MHGARP8 or LHG10, thespecified variable region and CDR sequences may comprise conservativesequence modifications. Conservative sequence modifications refer toamino acid modifications that do not significantly affect or alter thebinding characteristics of the antibody containing the amino acidsequence. Such conservative modifications include amino acidsubstitutions, additions and deletions. Modifications can be introducedinto an antibody of the invention by standard techniques known in theart, such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions are typically those in which anamino acid residue is replaced with an amino acid residue having a sidechain with similar physicochemical properties. Specified variable regionand CDR sequences may comprise one, two, three, four or more amino acidinsertions, deletions or substitutions. Where substitutions are made,preferred substitutions will be conservative modifications. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g. glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), beta-branched side chains (e.g. threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, one or more amino acidresidues within the CDR regions of an antibody of the invention can bereplaced with other amino acid residues from the same side chain familyand the altered antibody can be tested for retained function (i.e., theproperties set forth herein) using the assays described herein.anti-hGARP antibodies may also be CDR-grafted antibodies in which theCDRs are derived from a camelid antibody, for example a camelidanti-hGARP antibody raised by active immunization with hGARP.

In an embodiment, the invention provides an antibody that bindsessentially the same epitope as the MHGARP8 or LHG10 antibody.

In some embodiments of this invention, anti-hGARP antibodies comprisingVH and VL domains, or CDRs thereof may comprise CH1 domains and/or CLdomains, the amino acid sequence of which is fully or substantiallyhuman. Where the antigen binding polypeptide of the invention is anantibody intended for human therapeutic use, it is typical for theentire constant region of the antibody, or at least a part thereof, tohave a fully or substantially human amino acid sequence. Therefore, oneor more or any combination of the CH1 domain, hinge region, CH2 domain,CH3 domain and CL domain (and CH4 domain if present) may be fully orsubstantially human with respect to its amino acid sequence.Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain andCL domain (and CH4 domain if present) may all have a fully orsubstantially human amino acid sequence. In the context of the constantregion of a humanized or chimeric antibody, or an antibody fragment, theterm “substantially human” refers to an amino acid sequence identity ofat least 90%, or at least 95%, or at least 97%, or at least 99% with ahuman constant region. The term “human amino acid sequence” in thiscontext refers to an amino acid sequence which is encoded by a humanimmunoglobulin gene, which includes germline, rearranged and somaticallymutated genes. The invention also contemplates polypeptides comprisingconstant domains of “human” sequence which have been altered, by one ormore amino acid additions, deletions or substitutions with respect tothe human sequence, excepting those embodiments where the presence of a“fully human” hinge region is expressly required. The presence of a“fully human” hinge region in the anti-hGARP antibodies of the inventionmay be beneficial both to minimize immunogenicity and to optimizestability of the antibody. It is considered that one or more amino acidsubstitutions, insertions or deletions may be made within the constantregion of the heavy and/or the light chain, particularly within the Fcregion. Amino acid substitutions may result in replacement of thesubstituted amino acid with a different naturally occurring amino acid,or with a non-natural or modified amino acid. Other structuralmodifications are also permitted, such as for example changes inglycosylation pattern (e.g. by addition or deletion of N- or O-linkedglycosylation sites). Depending on the intended use of the antibody, itmay be desirable to modify the antibody of the invention with respect toits binding properties to Fc receptors, for example to modulate effectorfunction. For example cysteine residue(s) may be introduced in the Fcregion, thereby allowing interchain disulfide bond formation in thisregion. The homodimeric antibody thus generated may have improvedeffector function. See Caron et al., J. Exp. Med. 176: 1191-1195 (1992)and Shopes, B. J. Immunol. 148:2918-2922 (1992). Alternatively, a GARPantibody can be engineered which has dual Fc regions and may therebyhave enhanced complement lysis and ADCC capabilities. See Stevenson etal., Anti-Cancer Drug Design 3:219-230 (1989). The invention alsocontemplates immunoconjugates comprising an antibody as described hereinconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate). Fc regions may also be engineered for half-lifeextension, as described by Chan and Carter, 2010 Nature Reviews:Immunology, 10:301-316, incorporated herein by reference. Variantanti-hGARP antibodies in which the Fc region is modified by proteinengineering, as described herein, may also exhibit an improvement inefficacy (e.g. in therapeutics/diagnostics), as compared to anequivalent antibody (i.e. equivalent antigen-binding properties) withoutthe Fc modification.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. In still anotherembodiment, the glycosylation of an antibody is modified. For example,an aglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for the GARP target antigen. Suchcarbohydrate modifications can be accomplished by; for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Also envisaged arevariant anti-hGARP antibodies having an altered type of glycosylation,such as a hypofucosylated antibody having reduced amounts of fucosylresidues or a non-fucosylated antibody (as described by Natsume et al.,2009 Drug Design Development and Therapy, 3:7-16) or an antibody havingincreased bisecting GlcNac structures. Such altered glycosylationpatterns have been demonstrated to increase the ADCC activity ofantibodies, producing typically 10-fold enhancement of ADCC relative toan equivalent antibody comprising a “native” human Fc domain. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationenzymatic machinery (as described by Yamane-Ohnuki and Satoh, 2009 mAbs1(3):230-236).

In an embodiment of the invention, the anti-hGARP antibody comprises anFc region having the sequence SEQ ID NO: 47.

(SEQ ID NO: 47) PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment of the invention, the anti-hGARP antibodycomprises the heavy chain constant domain region having the sequence SEQID NO: 48, wherein X is N or is mutated into Q to inhibit ADCC.

(SEQ ID NO: 48) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYXSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In an embodiment of the invention, the residue 297 of SEQ ID NO: 48 isaglycosylated.

In another embodiment of the invention, the N residue at the position297 of SEQ ID NO: 48 is mutated into Q.

In an embodiment of the invention, the anti-hGARP antibody comprises thelight chain constant domain region having the sequence SEQ ID NO: 49.

(SEQ ID NO: 49) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC.

In further embodiments of the invention, anti-hGARP antibodies may belacking effector function, either because the Fc region of the antibodyis of an isotype which naturally lacks effector function, or whichexhibits significantly less potent effector function than human IgG1,for example human IgG2 or human IgG4, or because the Fc region of theantibody has been engineered to reduce or substantially eliminateeffector function, as described in Armour K L, et al., Eur. J. Immunol.,1999, 29:2613-2624.

In further embodiments, the Fc region of the anti-hGARP antibody may beengineered to facilitate the preferential formation of bispecificantibodies, in which two antibody heavy chains comprising differentvariable domains pair to form the Fc region of the bispecific antibody.Examples of such modifications include the “knobs-into-hole”modifications described by Ridgway J B, Presta L G, Carter P., 1996Protein Eng. July; 9(7):617-21 and Merchant. A M, et al. 1998 NatBiotechnol. July; 16(7):677-81.

In an embodiment of the invention, the anti-hGARP antibody of theinvention may exhibit one or more effector functions selected fromantibody-dependent cell-mediated cytotoxicity (ADCC), complementdependent cytotoxicity (CDC) and antibody-dependent cell-mediatedphagocytosis (ADCP) against cells expressing human GARP protein on thecell surface. The antibody may exhibit ADCC against GARP-relateddysfunctional cells. The antibody may exhibit enhanced ADCC function incomparison to a reference antibody which is an equivalent antibodycomprising a native human Fc domain. In a non-limiting embodiment, theADCC function may be at least 10× enhanced in comparison to thereference antibody comprising a native human Fc domain. In this context“equivalent” may be taken to mean that the antibody with enhanced ADCCfunction displays substantially identical antigen-binding specificityand/or shares identical amino acid sequence with the reference antibody,except for any modifications made (relative to native human Fc) for thepurposes of enhancing ADCC. The antibody may contain the hinge region,CH1 domain, CH2 domain and CH3 domain of a human IgG, most preferablyhuman IgG1. The antibody may include modifications in the Fc region,such as for example substitutions, deletions or insertion or otherstructural modifications to enhance or reduce Fc-dependentfunctionalities.

One object of this invention relates to anti-hGARP antibodies or antigenbinding fragment thereof which inhibit TGF-β signaling, and that may beparticularly suitable for therapeutic applications which benefit fromantibody effector function, i.e. ADCC, CDC, ADCP, and in particularenhanced effector function. Hence, the GARP antibodies described hereinwhich exhibit effector function (or enhanced effector function) andwhich inhibit TGF-β may be particularly advantageous for certaintherapeutic applications, e.g. cancer, chronic infection, and fibrosistreatments which benefit from antibody effector function.

Another object of the invention is an isolated polynucleotide sequenceencoding the heavy chain variable region of sequence SEQ ID NO: 8 or ofSEQ ID NO: 50. Preferably, said nucleic sequence is SEQ ID NO: 10:

ATGGCTGTCCTGGCATTACTCTTCTGCCTGGTAACATTCCCAAGCTGTATCCTTTCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACCGGCTATGGTATAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATGATATGGAGTGATGGAAGCACAGACTATAATTCAGTTCTCACATCCAGACTGAGGATCAGTAAGGATAATTCCAATAGCCAGGTTTTCTTAAAAATGAACAGTCTGCAAGTTGATGACACAGCCAGGTACTATTGTGCCAGAGATCGAAACTACTATGATTACGACGGGGCTATGGACTACTGGGGTCAAGGAA CCTCAGTCACCGTCTCCTCA.

Another object of the invention is an isolated polynucleotide sequenceencoding the light chain variable region of sequence SEQ ID NO: 9 or ofSEQ ID NO: 51. Preferably, said nucleic sequence is SEQ ID NO: 11:

ATGAAGTTTCCTTCTCAACTTCTGCTCTTCCTGCTGTTCAGAATCACAGGCATAATATGTGACATCCAGGTGACACAATCTTCATCCTACTTGTCTGTATCTCTAGGAGACAGGGTCACCATTACTTGCAAGGCAAGTGACCACATTAAAAATTGGTTAGCCTGGTATCAGCAGAAACCAGGAATTGCTCCTAGGCTCTTAGTTTCTGGTGCAACCAGTTTGGAAGCTGGGGTTCCTTCAAGATTCAGTGGCAGTGGATCTGGAAAGAATTTCACTCTCAGCATTACCAGTCTTCAGACTGAAGATGTTGCTACTTATTACTGTCAACAGTATTGGAGTACACCGTGGACGTTCGGTGGAGGCACCACTCTGGAGATCAGA.

Another object of the invention is an expression vector comprising thenucleic sequences encoding the anti-hGARP antibody of the invention. Inan embodiment, the expression vector of the invention comprises at leastone of SEQ ID NO: 10 and SEQ ID NO: 11 or any sequence having a nucleicacid sequence that shares at least: 60%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% identity with said SEQ ID NO: 10 and SEQ ID NO: 11.

Another object of the invention is an isolated host cell comprising saidvector. Said host cell may be used for the recombinant production of theantibodies of the invention. In an embodiment, host cells may beprokaryotic, yeast, or eukaryotic cells, and are preferably mammaliancells, such as, for example: monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen.Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); mouse myeloma cells SP2/0-AG14 (ATCC CRL1581; ATCC CRL 8287) or NSO (HPA culture collections no. 85110503);monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68(1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), aswell as DSM's PERC-6 cell line. Expression vectors suitable for use ineach of these host cells are also generally known in the art. It shouldbe noted that the term “host cell” generally refers to a cultured cellline. Whole human beings into which an expression vector encoding anantigen binding polypeptide according to the invention has beenintroduced are explicitly excluded from the definition of a “host cell”.

Another object of the invention is a method of producing an anti-hGARPantibody or antigen binding fragment thereof which comprises culturinghost cells containing the isolated polynucleotide sequence encoding theanti-hGARP antibody under conditions suitable for expression of theanti-hGARP antibody, and recovering the expressed anti-hGARP antibody.This recombinant process can be used for large scale production of GARPantibodies according to the invention, including antibodies monoclonalantibodies intended for in vitro, ex vivo, in vivo therapeutic,diagnostic uses. These processes are available in the art and will beknown by the skilled person.

Another object of the invention is a hybridoma cell line which can beused to produce said antibody of the invention.

A preferred hybridoma cell line according to the invention was depositedwith the BCCM/LMBP Plasmid Collection, Department of BiomedicalMolecular Biology, Ghent University, ‘Fiers-Schell-Van Montagu’building, Technologiepark 927, B-9052 Gent—Zwijnaarde BELGIUM (Table 2):

TABLE 2 Cell line Deposition No. Date of deposit MHGARP8 LMBP 10246CB 30May 2013 hybridoma

Fragments and derivatives of antibodies of this invention (which areencompassed by the term “antibody” or “antibodies” as used in thisapplication, unless otherwise stated or clearly contradicted bycontext), preferably a MHGARP8-like antibody, can be produced bytechniques that are known in the art. “Fragments” comprise a region ofthe intact antibody, generally the antigen binding site or variableregion. Examples of antibody fragments include Fab, Fab′, Fab′-SH,F(ab′)2, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single chain polypeptide”),including without limitation (1) single-chain Fv molecules (2) singlechain polypeptides containing only one light chain variable domain, or afragment thereof that contains the three CDRs of the light chainvariable domain, without an associated heavy chain moiety and (3) singlechain polypeptides containing only one heavy chain variable region, or afragment thereof containing the three CDRs of the heavy chain variableregion, without an associated light chain moiety; and multi-specificantibodies formed from antibody fragments. Fragments of the presentantibodies can be obtained using standard methods. For instance, Fab orF(ab′)2 fragments may be produced by protease digestion of the isolatedantibodies, according to conventional techniques. It will be appreciatedthat immune-reactive fragments can be modified using known methods, forexample to slow clearance in vivo and obtain a more desirablepharmacokinetic profile the fragment may be modified with polyethyleneglycol (PEG). Methods for coupling and site-specifically conjugating PEGto a Fab′ fragment are described in, for example, Leong et al, Cytokines16 (3): 106-119 (2001) and Delgado et al, Br. J. Cancer 73 (2): 175-182(1996), the disclosures of which are incorporated herein by reference.

Alternatively, the DNA of a hybridoma producing an antibody of theinvention, preferably a MHGARP8-like or LHG10-like antibody, may bemodified so as to encode a fragment of the invention. The modified DNAis then inserted into an expression vector and used to transform ortransfect an appropriate cell, which then expresses the desiredfragment.

In certain embodiments, the DNA of a hybridoma producing an antibody ofthis invention, preferably a MHGARP8-like or LHG10-like antibody, can bemodified prior to insertion into an expression vector, for example, bysubstituting the coding sequence for human heavy- and light-chainconstant domains in place of the homologous non-human sequences (e.g.,Morrison et al., PNAS pp. 6851 (1984)), or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies may be prepared that have the binding specificity of theoriginal antibody. Typically, such non-immunoglobulin polypeptides aresubstituted for the constant domains of an antibody of the invention.

Thus, according to another embodiment, the antibody of this invention,preferably a MHGARP8 or LHG10-like antibody, is humanized. “Humanized”forms of antibodies according to this invention are specific chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2, or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from the murine immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of theoriginal antibody (donor antibody) while maintaining the desiredspecificity, affinity, and capacity of the original antibody.

In some instances, Fv framework (FR) residues of the humanimmunoglobulin may be replaced by corresponding non-human residues.Furthermore, humanized antibodies can comprise residues that are notfound in either the recipient antibody or in the imported CDR orframework sequences. These modifications are made to further refine andoptimize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of the original antibody and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a region of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature, 321, pp.522 (1986); Reichmann et al, Nature, 332, pp. 323 (1988); Presta, Curr.Op. Struct. Biol., 3, pp. 394 (1992); Verhoeyen et al. Science, 239, pp.1534; and U.S. Pat. No. 4,816,567, the entire disclosures of which areherein incorporated by reference. Methods for humanizing the antibodiesof this invention are well known in the art.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of an antibody of this invention is screenedagainst the entire library of known human variable-domain sequences. Thehuman sequence that is closed to the mouse sequence is then accepted asthe human framework (FR) for the humanized antibody (Sims et al., J.Immunol. 151, pp. 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196, pp.901). Another method uses a particular framework from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework can be used for several differenthumanized antibodies (Carter et al., PNAS 89, pp. 4285 (1992); Presta etal. J. Immunol., 151 (1993)). It is further important that antibodies behumanized with retention of high affinity for GARP and other favorablebiological properties. To achieve this goal, according to a preferredmethod, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional structures ofselected candidate immunoglobulin sequences. Inspection of thesedisplays permits analysis of the likely role of the residues in thefunctioning of the candidate immunoglobulin sequence, i.e., the analysisof residues that influence the ability of the candidate immunoglobulinto bind its antigen. In this way, FR residues can be selected andcombined from the consensus and import sequences so that the desiredantibody characteristic, such as increased affinity for the targetantigen(s), is achieved. In general, the CDR residues are directly andmost substantially involved in influencing antigen binding. Anothermethod of making “humanized” monoclonal antibodies is to use a XenoMouse(Abgenix, Fremont, Calif.) as the mouse used for immunization. AXenoMouse is a murine host according to this invention that has had itsimmunoglobulin genes replaced by functional human immunoglobulin genes.Thus, antibodies produced by this mouse or in hybridomas made from the Bcells of this mouse, are already humanized. The XenoMouse is describedin U.S. Pat. No. 6,162,963, which is herein incorporated in its entiretyby reference.

Human antibodies may also be produced according to various othertechniques, such as by using, for immunization, other transgenic animalsthat have been engineered to express a human antibody repertoire(Jakobovitz et al. Nature 362 (1993) 255), or by selection of antibodyrepertoires using phage display methods. Such techniques are known tothe skilled person and can be implemented starting from monoclonalantibodies as disclosed in the present application.

In an embodiment, Camelidae hypervariable loops (or CDRs) may beobtained by active immunization of a species in the family Camelidaewith a desired target antigen. As discussed and exemplified in detailherein, following immunization of Camelidae (either the native animal ora transgenic animal engineered to express the immunoglobulin repertoireof a camelid species) with the target antigen, B cells producing(conventional Camelidae) antibodies having specificity for the desiredantigen can be identified and polynucleotide encoding the VH and VLdomains of such antibodies can be isolated using known techniques.

In an embodiment, the invention provides a recombinant antigen bindingpolypeptide immunoreactive with a target antigen, the polypeptidecomprising a VH domain and a VL domain, wherein at least onehypervariable loop or complementarity determining region in the VHdomain or the VL domain is obtained from a VH or VL domain of a speciesin the family Camelidae, which antigen binding polypeptide is obtainableby a process comprising the steps of:

-   -   (a) immunizing a species in the family Camelidae with a target        antigen or with a polynucleotide encoding said target antigen        and raising an antibody to said target antigen;    -   (b) determining the nucleotide sequence encoding at least one        hypervariable loop or complementarity determining region (CDR)        of the VH and/or the VL domain of a Camelidae conventional        antibody immunoreactive with said target antigen; and    -   (c) expressing an antigen binding polypeptide immunoreactive        with said target antigen, said antigen binding polypeptide        comprising a VH and a VL domain, wherein at least one        hypervariable loop or complementarity determining region (CDR)        of the VH domain or the VL domain has an amino acid sequence        encoded by the nucleotide sequence determined in part (a).

Isolated Camelidae VH and VL domains obtained by active immunization canbe used as a basis for engineering antigen binding polypeptidesaccording to the invention. Starting from intact Camelidae VH and VLdomains, it is possible to engineer one or more amino acidsubstitutions, insertions or deletions which depart from the startingCamelidae sequence.

In an embodiment, such substitutions, insertions or deletions may bepresent in the framework regions of the VH domain and/or the VL domain.The purpose of such changes in primary amino acid sequence may be toreduce presumably unfavourable properties (e.g. immunogenicity in ahuman host (so-called humanization), sites of potential productheterogeneity and or instability (glycosylation, deamidation,isomerization, etc.) or to enhance some other favourable property of themolecule (e.g. solubility, stability, bioavailability, etc.).

In another embodiment, changes in primary amino acid sequence can beengineered in one or more of the hypervariable loops (or CDRs) of aCamelidae VH and/or VL domain obtained by active immunization. Suchchanges may be introduced in order to enhance antigen binding affinityand/or specificity, or to reduce presumably unfavourable properties,e.g. immunogenicity in a human host (so-called humanization), sites ofpotential product heterogeneity and or instability, glycosylation,deamidation, isomerization, etc., or to enhance some other favourableproperty of the molecule, e.g. solubility, stability, bioavailability,etc.

The antibodies of the present invention, preferably a MHGARP8 orLHG10-like antibody, may also be derivatized to “chimeric” antibodies(immunoglobulins) in which a region of the heavy/light chain(s) isidentical with or homologous to corresponding sequences in the originalantibody, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity and binding specificity (Cabilly et al., supra;Morrison et al., Proc. Natl. Acad. Sci., pp. 6851 (1984)). An object ofthe invention is a composition comprising at least one of the protein ofthe invention as described here above.

Another object of the invention is a pharmaceutical compositioncomprising at least one of the protein of the invention as describedhere above and a pharmaceutically acceptable excipient.

Pharmaceutically acceptable excipients that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (for example sodiumcarboxymethylcellulose), polyethylene glycol, polyacrylates, waxes,polyethylene-polyoxypropylene- block polymers, polyethylene glycol andwool fat.

Another object of the invention is the protein of the invention forinhibiting TGF-β activity in a subject in need thereof.

Another object of the invention is a method for inhibiting TGF-βactivity in a subject in need thereof, comprising administering to thesubject an effective amount of the protein of the invention.

Another object of the invention is the protein of the invention or thepharmaceutical composition as defined here above for treating aTGF-β-related disorder in a subject in need thereof.

Another object of the invention is a method for treating a TGF-β-relateddisorder in a subject in need thereof, comprising administering to thesubject an effective amount of the protein of the invention.

Diseases or disorders where the methods of the invention can be usedinclude all diseases where inhibition of TGF-β can be beneficial.

Said TGF-β-related disorders include, but are not limited to,inflammatory diseases, chronic infection, cancer, fibrosis,cardiovascular diseases, cerebrovascular disease (e.g. ischemic stroke),and neurodegenerative diseases.

For use in administration to a subject, the composition will beformulated for administration to the subject. The compositions of thepresent invention may be administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term administration used herein includessubcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrastemal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques.

Sterile injectable forms of the compositions of this invention may beaqueous or an oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents that arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation.

Schedules and dosages for administration of the antibody in thepharmaceutical compositions of the present invention can be determinedin accordance with known methods for these products, for example usingthe manufacturers' instructions. For example, an antibody present in apharmaceutical composition of this invention can be supplied at aconcentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL)single-use vials. The product is formulated for intravenous (IV)administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citratedihydrate, 0.7 in g/mL polysorbate 80, and Sterile Water for Injection.The pH is adjusted to 6.5. It will be appreciated that these schedulesare exemplary and that an optimal schedule and regimen can be adaptedtaking into account the affinity and tolerability of the particularantibody in the pharmaceutical composition that must be determined inclinical trials.

Another object of the invention is a method for reducingimmunosuppression in the tumor environment in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of the protein of the invention.

Another object of the invention is a method for boosting the immunesystem in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of the protein of theinvention.

Another object of the invention is a method for inhibiting the immunesuppressive function of human Tregs in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of the protein of the invention.

Another object of the invention is a method for treating cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of the protein of the invention.

Another object of the invention is a method for treating cancer in asubject in need thereof, wherein the pharmaceutical composition of theinvention is to be administered as an immunostimulatory antibody fortreatment of cancer patients.

Another object of the invention is a method for treating cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of the protein of the invention incombination with another treatment for cancer or an immunotherapeuticagent.

Another object of the invention is a combination of the protein of theinvention and another treatment for cancer or another immunotherapeuticagent for treating or for use in treating cancer.

In an embodiment of the invention, said immunotherapeutic agent is atumor vaccine.

In another embodiment of the invention, said immunotherapeutic agent isan immunostimulatory antibody.

Without willing to be bound to a theory, the inventors believe theprotein of the invention will prevent immunosuppression in the tumorenvironment, thereby increasing the efficacy of the immunotherapeuticagent.

Various cancers can be treated by the present invention such as for anadrenocortical carcinoma, anal cancer, bladder cancer, brain tumor,glioma, breast carcinoma, carcinoid tumor, cervical cancer, coloncarcinoma, endometrial cancer, esophageal cancer, extrahepatic bile ductcancer, Ewing's tumor, extracranial germ cell tumor, eye cancer, gallbladder cancer, gastric cancer, germ cell tumor, gestationaltrophoblastic tumor, head and neck cancer, hypopharyngeal cancer, isletcell carcinoma, kidney cancer, laryngeal cancer, leukemia, lip and oralcavity cancer, liver cancer, lung cancer, lymphoma, melanoma,mesothelioma, merkel cell carcinoma, metastatic squamous head and neckcancer, myeloma, neoplasm, nasopharyngeal cancer, neuroblastoma, oralcancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreaticcancer, sinus and nasal cancer, parathyroid cancer, penile cancer,pheochromocytoma cancer, pituitary cancer, plasma cell neoplasm,prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell carcinoma,salivary gland cancer, skin cancer, Kaposi's sarcoma, T-cell lymphoma,soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, thyroidcancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer,or Wilms' tumor.

Suitable tumor antigens for use as a tumor vaccine known in the artinclude for example: (a) cancer-testis antigens such as NY-ESO-1, SSX2,SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, forexample, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6,and MAGE-12 (which can be used, for example, to address melanoma, lung,head and neck, NSCLC, breast, gastrointestinal, and bladder tumors), (b)mutated antigens, for example, p53 (associated with various solidtumors, e.g., colorectal, lung, head and neck cancer), p21/Ras(associated with, e.g., melanoma, pancreatic cancer and colorectalcancer), CD 4 (associated with, e.g., melanoma), MUM 1 (associated with,e.g., melanoma), caspase-8 (associated with, e.g., head and neckcancer), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701,beta catenin (associated with, e.g., melanoma), TCR (associated with,e.g., T-cell non-Hodgkins lymphoma), BCR-abl (associated with, e.g.,chronic myelogenous leukemia), triosephosphate isomerase, IA 0205,CDC-27, and LDLR-FUT, (c) over-expressed antigens, for example, Galectin4 (associated with, e.g., colorectal cancer), Galectin 9 (associatedwith, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g.,chronic myelogenous leukemia), WT 1 (associated with, e.g., variousleukemias), carbonic anhydrase (associated with, e.g., renal cancer),aldolase A (associated with, e.g., lung cancer), PRAME (associated with,e.g., melanoma), HER-2/neu (associated with, e.g., breast, colon, lungand ovarian cancer), alpha-fetoprotein (associated with, e.g.,hepatoma), SA (associated with, e.g., colorectal cancer), gastrin(associated with, e.g., pancreatic and gastric cancer), telomerasecatalytic protein, MUC-1 (associated with, e.g., breast and ovariancancer), G-250 (associated with, e.g., renal cell carcinoma), andcarcinoembryonic antigen (associated with, e.g., breast cancer, lungcancer, and cancers of the gastrointestinal tract such as colorectalcancer), (d) shared antigens, for example, melanoma-melanocytedifferentiation antigens such as MART-1/Melan A, gp100, MC1R,melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase relatedprotein-1/TRP1 and tyrosinase related protein-2/TRP2 (associated with,e.g., melanoma), (e) prostate associated antigens such as PAP, PSA,PSMA, PSH-P1, PSM-P1, PSM-P2, associated with e.g., prostate cancer, (f)immunoglobulin idiotypes (associated with myeloma and B cell lymphomas,for example), and (g) other tumor antigens, such as polypeptide- andsaccharide-containing antigens including (i) glycoproteins such assialyl Tn and sialyl Le<x> (associated with, e.g., breast and colorectalcancer) as well as various mucins; glycoproteins may be coupled to acarrier protein (e.g., MUC-1 may be coupled to LH); (ii)lipopolypeptides (e.g., MUC-1 linked to a lipid moiety); (iii)polysaccharides (e.g., Globo H synthetic hexasaccharide), which may becoupled to a carrier proteins (e.g., to KLH), (iv) gangliosides such asGM2, GM12, GD2, GD3 (associated with, e.g., brain, lung cancer,melanoma), which also may be coupled to carrier proteins (e.g., KLH).Other tumor antigens include pi 5, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET,IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, humanpapillomavirus (HPV) antigens, including E6 and E7, hepatitis B and Cvirus antigens, human T-cell lymphotropic virus antigens, TSP-180,p185erbB2, p180erbB-3, c-met, mn-23H 1, TAG-72-4, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, p 16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72,beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29BCAA), CA 195, CA 242,CA-50, CAM43, CD68KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344,MA-50, MG7-Ag, MOV 18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP,TPS, and the like.

Suitable immunostimulatory antibodies include, but are not limited to:anti-CTLA-4, anti-PD1, anti-PDL1 and anti-KIR antibodies.

In an embodiment of the invention, the method for treating cancer in asubject in need thereof, comprises administering to the subject theprotein of the invention prior to, concurrent to and/or posterior toanother anti-cancer agent or cancer treatment, such as chemotherapytreatment.

Another object of the present invention is a method to preventinfectious diseases such as HIV, malaria, or Ebola, or improvevaccination against these infections, comprising administering to thesubject a therapeutically effective amount of the protein of theinvention.

In an embodiment, the protein of the invention may be used in vitro orin vivo to identify samples, tissues, organs or cells that express GARP.

Examples of assays in which the protein of the invention may be used,include, but are not limited to, ELISA, sandwich ELISA, RIA, FACS,tissue immunohistochemistry, Western-blot, and immunoprecipitation.

In an embodiment of the invention, the sample is a biological sample.Examples of biological samples include, but are not limited to, bodilyfluids, preferably blood, more preferably blood serum, plasma, synovialfluid, bronchoalveolar lavage fluid, sputum, lymph, ascitic fluids,urine, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleuralfluid, pericardial fluid, and alveolar macrophages, tissue lysates andextracts prepared from diseased tissues.

In an embodiment of the invention, the term “sample” is intended to meana sample taken from an individual prior to any analysis.

In another embodiment, the protein of the invention may be labeled fordiagnostic or detection purposes. By labeled herein is meant that acompound has at least one element, isotope or chemical compoundsattached to enable the detection of the compound. Examples of labelsinclude, but are not limited to, isotopic labels such as radioactive orheavy isotopes; magnetic, electric or thermal labels and colored orluminescent dyes. For example: lanthanide complexes, quantum dots,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, malachite green, stilbene, Luciferyellow, cascade blue, texas red, alexa dyes, cy dyes.

One object of the invention is a method for identifying activated Tregsin a sample based on the use of the protein of the invention.

Another object of the invention is a method for identifying soluble orcomplexed latent TGF-β based on the use of the protein of the invention.

Another object of the invention is a kit comprising at least one proteinof the invention.

By “kit” is intended any manufacture (e.g., a package or a container)comprising at least one reagent, i.e. for example an antibody, forspecifically detecting the expression of GARP. The kit may be promoted,distributed, or sold as a unit for performing the methods of the presentinvention. Furthermore, any or all of the kit reagents may be providedwithin containers that protect them from the external environment, suchas in sealed containers. The kits may also contain a package insertdescribing the kit and methods for its use.

Kits for performing the sandwich ELISA methods of the inventiongenerally comprise a capture antibody, optionally immobilized on a solidsupport (e.g., a microtiter plate), and a revelation antibody coupledwith a detectable substance, such as, for example HRP, a fluorescentlabel, a radioisotope, beta-galactosidase, and alkaline phosphatase.

EXAMPLES

The present invention is further illustrated by the following examples.

Example 1: New Monoclonal Antibodies Directed Against Human GARP(Anti-hGARP Monoclonals)

DBA/2 or Balb/c mice were immunized with murine P1HTR cells transfectedwith human GARP. Sera from immunized mice were tested for the presenceof anti-hGARP antibodies, by screening for binding to hGARP-expressingBW cells by FACS. Splenocytes from mice with high titers of anti-hGARPantibodies were fused to SP2/neo cells. Hybridomas were selected in HATmedium and cloned under limiting dilution. Supernatants of +/−1600hybridoma clones were screened by FACS for the presence of antibodiesbinding to hGARP-expressing BW cells. Thirty-eight clones producinganti-hGARP monoclonal antibodies were identified in this screening. Nineclones were selected and amplified for large scale-production andpurification of nine new anti-hGARP monoclonals (MHGARP1 to 9).

As shown in FIG. 1, MHGARP1 to 9 bind to murine BW5147 cells transfectedwith hGARP, but not to untransfected cells. MHGARP1 to 9 also bind 293Tcells transfected with hGARP and two human T cells lines (clone Th A2and Jurkat) transduced with a hGARP-encoding lentivirus, but not thecorresponding parental cells (not shown). This recognition pattern isidentical to that of a commercially available anti-hGARP mAb (clonePlato-1) used here as a positive control. These results show thatMHGARP1 to 9 recognize hGARP on cell surfaces.

As shown in FIGS. 7A-7B, five additional MHGARP antibodies were producedand purified. MHGARP antibodies (MHG-1 to -14 on the figure) do not bindclone ThA2 (human CD4+ T helper cells, which do not express hGARP), butbind ThA2 transduced with hGARP.

Example 2: MHGARP8, but None of 12 Other Anti-hGARP Monoclonals,Inhibits Active TGF-β Production by Human Treg Cells

A human Treg clone (1E+06 cells/ml) was stimulated in serum-free mediumwith coated anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml)antibodies, in the presence or absence of 20 μg/ml of an anti-hGARPmonoclonal antibody. Thirteen anti-hGARP monoclonals were tested in thisassay: the above mentioned nine new monoclonals (MHGARP1 to 9), andcommercially available antibody clones Plato-1 (Enzo Life Sciences,catalog No. ALX-804-867), 272G6 (Synaptic Systems, catalog No. 221 111),50G10 (Synaptic Systems, catalog No. 221 011) and 7B11 (BioLegend,catalog No. 352501). Cells were collected after 24 hours, lysed andsubmitted to SDS-PAGE under reducing conditions. Gels were blotted onnitrocellulose membranes with the iBlot system (Life Technologies).After blocking, membranes were hybridized with primary antibodiesdirected against phosphorylated SMAD2 (pSMAD2, Cell SignalingTechnologies) or β-ACTIN (SIGMA), then hybridized with secondaryHRP-coupled antibodies and revealed with Enhanced ChemiLuminescent (ECL)substrate (ThermoFisher Scientific). The presence of pSMAD2 indicatesproduction of active TGF-β1 by the stimulated Treg clone. ECL signalswere quantified by measuring the density of the 55 kDa pSMAD2 and 40 kDaβ-ACTIN bands on autoradiographs, using the Image J software.

To examine whether hGARP is required for active TGF-β production byTCR-stimulated Treg cells, a human Treg clone was stimulated through itsT cell receptor (TCR), alone or in the presence of anti-hGARP mAbs.Active TGF-β produced by stimulated Tregs triggers an autocrine signal,which leads to the phosphorylation and activation of SMAD2 and SMAD3transcription factors. The presence of phosphorylated SMAD2 (pSMAD2) wasmeasured by Western Blot (WB) as read-out for active TGF-β production bythe stimulated Treg clone. As shown in FIGS. 2A-2B, pSMAD2 was reducedmore than 10 fold in the presence of MHGARP8. This reduction is similarto that observed in the presence of an anti-TGF-β mAb, used here as apositive control. None of the twelve other anti-hGARP mAbs (eight otherin-house produced MHGARP and four commercially available anti-GARPantibodies) inhibited active TGF-β production by the Treg clone.Altogether, our data demonstrate that GARP is required for active TGF-βproduction by human Tregs, as MHGARP8, an antibody directed againsthGARP, prevented active TGF-β production.

Example 3: MHGARP8, But not Other Anti-hGARP mAbs, Recognizes aConformational Epitope that Requires the Presence of TGF-β

Mapping the regions recognized by anti-hGARP monoclonals Murine BW5147 Tcells were electroporated with plasmids encoding the HA-tagged proteinsschematized in FIG. 3A, corresponding to hGARP, mGARP or mGARP/hGARPchimeras. Stable clones selected in neomycin were stained withbiotinylated anti-hGARP antibodies (anti-hGARP1 to 9) andstreptavidin-PE, with the commercial anti-hGARP antibody (clone Plato-1)and a secondary anti-mIgG2b-AF488, or with an anti-HA antibody andsecondary anti-mouse IgG1-AF488. Histograms are gated on live cells.Black histograms show signals on untransfected BW cells, whitehistograms show signals on BW cells expressing the HA-tagged hGARP, andgrey histograms show signals on BW cells expressing HA-tagged mGARP ormGARP/hGARP chimeras.

Parental BW5147 T cells (BW non-transfected) or clones stablytransfected with hGARP alone (BW+hGARP) or with hTGFB1(BW+hGARP+hTGF-β1) were stained with biotinylated anti-hGARP antibodies(anti-hGARP1 to 9) and streptavidin-PE, with the commercial anti-hGARPantibody (clone Plato-1) and a secondary anti-mIgG2b-AF488, or withanti-mLAP-AF647 or anti-hLAP-APC antibodies.

The mechanism by which MHGARP8, but not other anti-hGARP mAbs, inhibitsactive TGF-β production by Tregs was investigated. It was hypothesizedthat MHGARP8 may recognize an epitope in hGARP that is distinct from theepitopes recognized by the other anti-hGARP mAbs.

With the exception of MHGARP-1, the MHGARP mAbs of the instantapplication do not recognize murine GARP (mGARP). Plasmids weretherefore constructed encoding HA-tagged hGARP, mGARP or hGARP/mGARPchimeras to map the hGARP regions recognized by the mAbs of the instantapplication. Murine BW cells were transfected and stable clones werederived expressing the HA-tagged proteins (schematically represented inFIGS. 3A-3E). All clones expressed similar levels of HA-tagged proteinon the surface, as indicated by similar fluorescence intensities afterstaining with an anti-HA mAb (FIG. 3A). As expected, all the MHGARP mAbsbound to the clone expressing HA-tagged hGARP, whereas none, exceptMHGARP-1, bound to the clone expressing HA-tagged mGARP. Four groups ofmAbs emerged from the analysis of binding to the HA-tagged hGARP/mGARPchimeras (FIG. 3A). Monoclonal antibodies in the first group (MHGARP-6,-7 and -9) bound none of the chimeras, indicating that they recognize anepitope located between aa 20 and 101 of hGARP (region 20-101). mAbs inthe second group (MHGARP-2, -3 and -8) bound to only 1 of the 5chimeras, and thus recognize an epitope in region 101-141. A third groupcomprises MHGARP-5, which bound to 2 of the chimeras and thereforerecognizes region 141-207. This group probably also contains MHGARP-1,which is cross-reactive but bound these 2 chimeras more efficiently thanit bound mGARP or the 3 other chimeras. Finally, mAbs in the fourthgroup (MHGARP-4 and Plato-1) bound 4 of the 5 chimeras, and thusrecognize region 265-333.

Based on the above, the anti-hGARP mAbs were grouped into four familiesof antibodies that recognize four distinct regions of the hGARP protein.MHGARP-8, which displays neutralizing activity, binds to region 101-141.This region is also recognized by MHGARP-2 and -3, which are notneutralizing. Therefore, the ability to bind region 101-141 is notsufficient to confer neutralizing activity.

To further define the epitopes recognized by MHGARP-2, -3 and -8, thebinding of the anti-hGARP antibodies was compared to clones of BW cellsexpressing hGARP alone (BW+hGARP), or hGARP and hTGF-β1(BW+hGARP+hTGF-β1). With the notable exception of MHGARP8, allanti-hGARP antibodies stained BW+hGARP+hTGF-β1 with the same intensityas BW+hGARP, indicating that the two clones express the same levels ofhGARP on the cell surface. The MHGARP8 antibody in contrast, stainedBW+hGARP+hTGF-β1 more intensely than BW+hGARP (FIG. 3B). This indicatesthat although hGARP levels are similar on the two clones, the epitoperecognized by MHGARP8 is more abundant on BW+hGARP+hTGF-β1 than onBW+hGARP cells.

A plausible explanation for this observation is that the epitoperecognized by MHGARP8 appears only when hGARP is bound to murine (m) orhuman (h) TGF-β1. This could be due to one of two mechanisms: either theepitope comprises amino-acids from both hGARP and TGF-β1 (mixedconformational epitope), or it comprises amino-acids from hGARP only,but that adopt a different conformation in the presence of TGF-β1(binding-induced conformational epitope). BW cells express murineTGF-β1, and murine TGF-β1 binds to hGARP (FIG. 3B). Therefore, bindingof MHGARP8 to BW+hGARP (in the absence of transfected hTGF-β1) could bedue to recognition of hGARP/mTGF-β1 complexes.

To explore the hypothesis that MHGARP8 recognizes GARP when it is boundto TGF-β1, co-immunoprecipitation experiments were performed. Thedifferent anti-GARP antibodies were used to immunoprecipitate GARP fromBW+hGARP+hTGF-β1 cells, then analyzed to determine if TGF-β wasco-immunoprecipitated with GARP. As shown in FIG. 3C, all anti-GARPantibodies efficiently immunoprecipitated GARP (FIG. 3C, top panels).Co-immunoprecipitation of TGF-β1 was observed with MHGARP-6, -7, -8, and-9 mAbs, indicating that these antibodies bind GARP bound to TGF-β1. Incontrast, MHGARP-1, -2, -3, -4 and -5 immunoprecipitated GARP asefficiently as the other anti-GARP mAbs, but they did notco-immunoprecipitate TGF-β (FIG. 3C, bottom panels). This indicates thatMHGARP-1, -2, -3, -4 and -5 recognize free GARP, but not GARP that isbound to TGF-β. It is important to note that MHGARP-2 and -3, whichrequire the GARP₁₀₁₋₁₄₁ region for binding, recognize only free GARP,whereas neutralizing MHGARP8, which also requires GARP₁₀₁₋₁₄₁,recognizes GARP bound to TGF-β.

To confirm this observation, 293T cells, which express low levels ofendogenous TGF-β1, were used to co-transfect hGARP with increasingamounts of hTGFB1 (FIG. 3D). Binding of MHGARP-1, -2, -3, -4 and -5decreased dose-dependently when hTGFB1 was co-transfected with hGARP. Itwas completely abrogated at the highest doses of hTGFB1. This confirmsthat MHGARP-1 to -5 bind only free GARP. Binding of MHGARP-6, -7, and -9was not modified by co-transfection of hTGFB1, indicating that thesemAbs bind hGARP whether or not it is bound to TGF-β1 (i.e. they bindboth free GARP and GARP bound to TGF-β1). In striking contrast, bindingof MHGARP8 increased dose-dependently when hTGFB1 was co-transfectedwith hGARP. This suggests again that in contrast to all otherantibodies, MHGARP8 does not bind free GARP, but only GARP bound toTGF-β1.

To demonstrate that MHGARP8 binding requires the presence of TGF-β1,siRNAs were used to silence the expression of TGFB1 in Jurkat cellstransduced with hGARP (FIG. 3E). The siRNA against TGFB1 mRNAefficiently reduced expression of TGF-β1, as illustrated by the decreasein surface LAP detected on Jurkat+hGARP cells (FIG. 3E, right panel).Reduced expression of TGF-β1 in Jurkat+hGARP decreased the binding ofthe MHGARP8 antibody, but it did not modify the binding of the otheranti-GARP antibodies (FIG. 3E, foreground histograms). This confirmsthat in contrast to the other anti-GARP antibodies, MHGARP8, does notbind free GARP, but only binds GARP in the presence of TGF-β1.

Finally, the unlikely hypothesis that presentation of TGF-β on the cellsurface, irrespective of hGARP expression, is sufficient for binding byMHGARP8, was evaluated. In other words, whether MHGARP8 recognizes amixed or a binding-induced conformational epitope that requiresexpression of both hGARP and TGF-β was analyzed. For this, 293T cellswere transfected with constructs encoding hGARP, mGARP or thehGARP/mGARP chimeras described above, with or without a constructencoding hTGF-β1. Transfected cells were analyzed by FACS to measurebinding of the MHGARP8 antibody, and presentation of hTGF-β1 on the cellsurface with an anti-hLAP antibody (FIGS. 4A-4B). By comparison tounstransfected cells, transfection of hGARP, mGARP or hGARP/mGARPconstructs alone (no hTGFB1) induced low levels of surface LAP, due tolow levels of endogenous hTGFB1 expression (FIG. 4A, left). Surface LAPlevels dramatically increased upon transfection of hTGFB1 in cellstransfected with hGARP, mGARP, or any hGARP/mGARP construct (FIG. 4B,left histogram). This indicates that hTGF-β1 is presented on the cellsurface by hGARP, by mGARP and by all the hGARP/mGARP chimeras.Importantly, MHGARP8 bound only to the surface of cells transfected withhGARP, or with the hGARP/mGARP constructs encoding amino-acids 101 to141 of hGARP (FIGS. 4A-4B, right). It did not bind to cells transfectedwith hTGFB1 and mGARP, nor to cells transfected with hTGFB1 andhGARP/mGARP constructs that do not encode hGARP101-141 (FIG. 4B, right),although these cells presented high levels of LAP on their surface (FIG.4B, left). This demonstrates that presentation of TGF-β1 on the cellsurface (by mGARP or hGARP/mGARP chimeras) is not sufficient for bindingby MHGARP8. Binding of MHGARP8 requires the presence of both hGARP(region 101-141) and TGF-β1 on the cell surface.

As indicated above, MHGARP8 does not bind mGARP. Its binding to hGARPrequires a region comprising amino-acids 101 to 141. To further definethe epitope recognized by MHGARP8, the sequences of region 101-141 inhuman and murine GARP were compared. In this region, only 13 amino-acidsdiffer between hGARP and mGARP (FIG. 5, amino-acids highlighted by greyboxes). Three HA-tagged mutant forms of hGARP were constructed. In eachmutant (Mut I, Mut II and Mut III), three consecutive amino-acids werereplaced by the corresponding amino-acids of the mGARP protein (FIG. 5,black boxes). Stable clones of BW cells transfected with these HA-taggedforms of wild type (WT) or mutant hGARP were derived. All clonesexpressed similar levels of HA-tagged protein on the surface, asdemonstrated by staining with an anti-HA antibody (FIG. 5, histograms onthe right). The clones were then analyzed after staining with MHGARP-2,-3 and -8, i.e., antibodies which require region 101-141 of hGARP forbinding. The three antibodies bound to cells expressing WT, Mut I andMut II forms of hGARP. In contrast, binding was greatly reduced on cellsexpressing the Mut III form of hGARP, indicating that MHGARP-2, -3 and-8 require amino-acids 137-138-139 of hGARP for binding.

Altogether, the data show that MHGARP8 is the only available anti-GARPantibody that inhibits active TGF-β1 production by human Tregs. Thisneutralizing activity is linked to binding of MHGARP8 to an epitope thatis distinct from those bound by all other anti-GARP antibodies: bindingof MHGARP8 requires both region 101-141 of hGARP and the presence ofhTGF-β, whereas binding of non-neutralizing antibodies require otherregions of hGARP (for MHGARP-1, -4, -5, -6, -7 and -9), or occurs onlyin the absence of TGF-β1 (for MHGARP-2 and -3). In region hGARP101-141,amino-acids 137 to 139 are required for the binding of MHGARP-2, -3 and-8.

The affinity of the MHGARP8 antibody to immobilized shGARP-TGFβ wasmeasured by BIACOR analysis. The Kd of said antibody is 0.2 nM.

Example 4: MHGARP8 Inhibits Human Treg Cell Function In Vivo

To examine whether MHGARP8 also inhibits human Tregs in vivo, a model ofxenogeneic GvHD induced by transfer of human PBMCs (Peripheral BloodMononuclear Cells) into immuno-compromised NOD-Scid-IL2Rg^(−/−) (NSG)mice was used. NSG mice lack functional T, B and NK cells. This allowsefficient engraftment of human hematopoietic stem cells (HSCs), whichproliferate and generate a functional human immune system in recipientmice. When human PBMCs are used instead of HSCs, efficient engraftmentof T cells occurs, but is soon accompanied by the development of axenogeneic Graft-versus-Host Disease (GvHD). In this model, GvHD resultsfrom the activity of human donor cytotoxic T lymphocytes that recognizetissues of the recipient NSG mice as foreign (Shultz, et al. Nature2012, 12:786-798). The severity of the GvHD can be decreased byco-transferring human Treg cells with human PBMCs (Hannon et al.Transfusion 2014).

Human PBMCs were isolated from total blood of a hemochromatosis donor bycentrifugation on density gradients (Lymphoprep™), and frozen for lateruse. Autologous Tregs were generated as follows: CD4+ T cells wereisolated from the blood of the same donor using the RosetteSep™ HumanCD4+ T Cell Enrichment Cocktail (StemCell Technologies) and stained withanti-CD4, anti-CD25 and anti-CD127 antibodies coupled to fluorochromes.CD4+CD25hiCD127lo cells were sorted by flow cytometry (>99% purity) thenstimulated with anti-CD3/CD28 coated beads (Dynabeads® Human T-ActivatorCD3/CD28 for T-Cell Expansion and Activation, Life Technologies) in thepresence of IL-2 (120 IU/ml) during 14 days. These expanded Treg cellswere frozen for later use.

NSG mice were irradiated (2.5 Gy) on day −1, then injected in the tailvein with human PBMCs (2.7×106 per mouse) alone, or mixed with expandedhuman Tregs (1.4×106 per mouse) on day 0. Mice also received weekly i.p.injections of MHGARP8 antibody (400 μg on day −1 (day minus 1), 200 μgat later time points), or control PBS. Mice were monitored bi-weekly forGvHD development as indicated in the text.

Human PBMCs with or without Tregs were transferred into NSG mice, andthe mice were treated with i.p. injections of MHGARP8 antibody orcontrol PBS. The large number of human Treg cells required for thetransfers were obtained through short in vitro amplification ofCD4+CD25+CD127lo cells sorted from human PBMCs by flow cytometry.Objective signs of GvHD development in the recipient mice were monitoredbi-weekly. We performed two independent experiments, which yieldedsimilar results. In experiment 1 (FIG. 6A), signs of GvHD (meanscore >1) appeared 29 days after injection of human PBMCs (group I;n=2). Disease severity increased quickly, and one of the 2 mice waseuthanized for ethical reasons on day 55. In mice injected with PBMCsand Tregs (group II; n=3), the appearance of GvHD was delayed bycomparison to PBMCs alone (mean score >1 reached after 58 days). Thisindicates that Tregs, as expected, partially protected NSG mice againstGvHD. Importantly, treatment of mice receiving PBMCs and Tregs with theMHGARP8 antibody (group III, n=6) aggravated the disease: signs of GvHDappeared earlier (36 days) than in mice from group II. The effect ofMHGARP8 appears to depend on the presence of Tregs, as no difference indisease score was observed between mice receiving PBMCs only (group I)or PBMCs and MHGARP8 (group IV; n=4). This experiment was repeated witha larger number of mice per group (FIG. 6B). Again, co-injection ofTregs with PBMCs delayed the appearance of GvHD by comparison to PBMCsalone (day 46 in group II versus day 28 in group I), and treatment withthe MHGARP8 antibody aggravated GvHD in mice receiving PBMCs and Tregs(day 28 in group III) by comparisons to untreated mice (day 46 in groupII). Altogether, this shows that MHGARP8 inhibits the immune-suppressivefunction of human Tregs in vivo.

Example 5: New Anti-hGARP Monoclonal Antibodies (mAbs) UsingImmunization of Llamas Approach

Production of Recombinant Soluble GARP-TGFβ1 Complex

Human and murine GARP-TGFβ1 complex was produced as a soluble complexusing a truncated GARP expression construct. The human GARP proteinsequence was truncated after Leucine 628, followed by a cleavable TEV-3×strep tag (EAAENLYFQGAAWSHPQFEKGAAWSHPQFEKGAAWSHPQFEKGAA*) (SEQ ID NO:40). Murine GARP protein sequence was truncated after leucine 629,followed by the same cleavable TEV-3× strep tag. The GARP-TGFβ1complexes were produced by co-expression of the truncated GARP and theTGFβ1 in HEK293E cells, followed by purification via the Strep-Tag.

Immunization of Llamas

Immunizations of llamas and harvesting of peripheral blood lymphocytes(PBLs) as well as the subsequent extraction of RNA and amplification ofantibody fragments were performed as described by De Haard andcolleagues (De Haard H, et al., J. Bact. 187:4531-4541, 2005). Fourllamas were immunized with BW cells over-expressing human GARP and TGFβ1(FIG. 7A) as confirmed by flow cytometry using MHGARP8 (MHG-8)monoclonal antibody described in this patent application. The llamaswere immunized with intramuscular injections in the neck once per weekfor a period of six weeks. Approximately 10⁷ cells were injected intothe neck muscles and Freund's incomplete adjuvant was injected in asecond region located a few centimeters from the injection site of thecells. Another four llamas were immunized with a mix of human GARP cDNAand human TGFβ1 cDNA expression vectors, once per two weeks, with fourrepetitive injections.

Blood samples of 10 ml were collected pre- and post-immunization toinvestigate the immune response. Three to four days after the lastimmunization, 400 ml of blood was collected for extraction of total RNAfrom the PBLs prepared using a Ficoll-Paque gradient and the methoddescribed by Chomczynski P, et al., Anal. Biochem. 162: 156-159, 1987.On average, RNA yields of 450 μg were achieved, which was used forrandom cDNA synthesis and PCR amplification of the V-regions of theheavy and the light chains (Vλ and Vκ) for construction of the Fabcontaining phagemid libraries as described by De Haard H et al., (J BiolChem. 1999 Jun. 25; 274(26): 18218-30), to obtain diverse libraries ofgood diversity (1-7×10⁸).

The immune response to the GARP-TGF β1 complex was investigated by ELISAon coated recombinant soluble GARP-TGF β1 complex (1 μg/ml). Five-foldserial dilutions of sera, starting from 10% sera were prepared and 100μl of diluted sera was added onto the coated wells and incubated for 1hour at RT. After washing with 3×PBS/Tween, the plates were blocked withPBS supplemented with 1% casein (FIGS. 8A-8B). Binding of conventionalllama IgG1 to its target GARP-TGFβ was measured in ELISA using a mouseanti llama IgG1 antibody (clone 27E10, Daley L P, et al. Clin. Diagn LabImmunol. 12, 2005) and a HRP-conjugated donkey anti-mouse antibody(Jackson) for detection.

Selections and Screenings of GARP-TGFβ1 Specific Fabs

Phage expressing Fabs were produced according to standard protocols andselections performed on immobilized recombinant soluble GARP-TGFβ1 withtotal elution of the GARP-TGFβ1 binding phage with trypsin according tostandard phage display protocols.

Two to three rounds of selections were performed to enrich for humanGARP-TGFβ1 specific Fabs expressed by the phage. hGARP and hTGFβ1 (LAP)counter selections were used to enrich for Fabs binding the hGARP-TGFβ1complexes. Individual colonies were isolated and periplasmic fractions(peris) in 96-well plates were produced by IPTG induction from all thelibraries according to standard protocols.

Screening of the hGARP-TGFβ specific Fabs was performed using ELISA.hGARP-TGFβ1 was immobilized on a maxisorb plate. After blocking with 1%casein in PBS for 1 h, Fab from 20 μl periplasmic extracts were allowedto bind to hGARP-TGFβ1.

Characterization of Monoclonal Antibodies

GARP-TGFβ1/GARP specific clones were sequenced in the VH and the VLregions and divided into VH families based on the sequence of the CDR3in the VH. Seventeen families were identified. Of each VH familyidentified at least one representative clone was cloned into a fullhuman IgG1 (LHG1-LHG17). These monoclonal antibodies were analyzed onBiacore for their binding characteristics to soluble human GARP-TGFβ1complex. Recombinant soluble human GARP-TGFβ1 was immobilized atapproximately 4,000 RU on a CM5 chip (GE Healthcare).

Binding of monoclonal antibodies to the human and cynomolgus GARP-TGFβ1complex expressed on HEK-293 cells was analyzed by FACS. Cynomolgus GARPand cynomolgus TGFβ1 encoding sequences were cloned from a cDNA samplefrom cynomolgus peripheral blood lymphocytes (PBMCs). Primers were basedon the predicted sequences of cynomolgus GARP (XM_005579140.1; SEQ IDNO: 41) and cynomolgus TGFβ1 (XM_005589338.1; SEQ ID NO: 42) byamplification of overlapping parts of the full sequence. For bothcynomolgus GARP and cynomolgus TGFβ1 three separate PCR amplicons wereDNA sequence analyzed. They fully aligned with the predicted sequences.Cynomolgus GARP and cynomolgus TGFβ1 were cloned into pCDNA3.1 fortransient over-expression in HEK293E cells. Binding to cynomolgusGARP-TGFβ1 was compared to binding to human GARP-TGFβ1 on FACS. LHG-10and the shuffled variants (LHG-10.3 to LHG-10.6) can be considered ascross-reactive with cynomolgus GARP-TGFβ1 (FIG. 9).

Primers Used:

>cyno TGFB S1: (SEQ ID NO: 43) cgcctc CCCCATGCCG ccctccg >cyno TGFB S2:(SEQ ID NO: 44) acaattcctg gcgatacctc >cyno TGFB AS1: (SEQ ID NO: 45)CTCAACCACTGCCGCACAAC >cyno TGFB AS2: (SEQ ID NO: 46)TCAGCTGCATTTGCAGGAGC

VK Shuffling for Improved Affinity

VK chain shuffling was used to improve the affinity of the mAb LHG-10(FIG. 10). In this method, the heavy chain of the parental clone (VHCH1of LHG-10) was reintroduced in the phagemid-light chain library. Theheavy chain was extracted from an expression vector, which lacks thebacteriophage derived gene 3 necessary for display, to further avoidcontamination of the parental light chain in the selection procedure.The heavy chain was cloned into the phagemid-light chain library and theligated DNA was electroporated into E. coli TG1 cells to create thelight chain shuffled library. The size of libraries was above 10⁸.

Affinity selections, combined with off-rate washes, were performed toselect for chain shuffled Fabs with an improved affinity for humanGARP-TGFβ1. A set-up was chosen where Fab expressing phages wereincubated with different concentrations of recombinant soluble humanGARP-TGFβ1 directly coated to the microsorb plate.

By adding the recombinant soluble human GARP-TGFβ1 in excess over thecoated recombinant soluble human GARP-TGFβ1, the binding of the higheraffinity phage was favored. Each round the time of washing was increased(Table 3) to select for phages with a better off-rate by washing awaythe lower affinity variants. Phages were eluted with trypsin and usedfor infection of E. coli TG1 cells. In total, five rounds of selectionwere done. In addition the amount of input phage was decreased insubsequent rounds to reduce background on the one hand and on the otherhand to lower the mAb concentration thereby increasing the stringency ofthe selection.

TABLE 3 Parameters varied for each round of selection for VK shufflingRI RII RIII RIV RV Concentrations 10 μg/ml 10 μg/ml 10 μg/ml 10 μg/ml 10μg/ml rhGARP-TGFβ 1 μg/ml 1 μg/ml 1 μg/ml 1 μg/ml 1 μg/ml 0.1 μg/ml 0.1μg/ml 0.1 μg/ml 0.1 μg/ml 0.1 μg/ml Vol. Phage 10 μl 1 μl 1 μl 1 μl 1 μlTime of 0 h 2 h O/N O/3N O/6N washing Conditions — 37° C., 37° C., 37°C., 37° C., 100 μg/ml 100 μg/ml 100 μg/ml 100 μg/ml rhGARP-TGFβ rhGARP-rhGARP- rhGARP- in 1% casein TGFβ TGFβ TGFβ in 1% casein in 1% casein in1% casein

Screenings of at least 24 clones from selection rounds III, IV and Vwere performed. The clones were grown in deep well plates (1 mlexpressions) and periplasmic fractions were prepared. These periplasmicextracts were analyzed on Biacore for improved off-rates. Top four Fabclones with improved off-rates were cloned into hIgG1 (LHG-10 series)and also an effector-dead variant hIgG1 with an N297Q substitution inthe Fc region (LHG-10-D series), and the resultant IgGs were analyzedfor improved binding characteristics on Biacore (Table 4). In addition,the LHG-10-D IgGs were checked for cross-reactivity on cyno GARP/cynoTGF-β1 in a FACS-based assay using HEK-293E cells transfected with cynoGARP/cyno TGFβ1 or human GARP/human TGFβ1. MHGARP8 was also tested inthis cross-reactivity assay. All LHG-10-D and MHG-8 are cross-reactiveagainst cyno GARP/cyno TGFβ1 (FIG. 9).

TABLE 4 Binding characteristics of shuffled clones fold Fold fold ka(1/Ms) improvement kd (1/s) improvement KD improvement MHGARP8 1.25E+05N/A 3.39E−05 N/A 2.64E−10 N/A LHG- 1.42E+05 1.0 2.62E−05 1.0 1.85E−101.0 10-D LHG- 2.31E+05 0.6 5.18E−06 5.1 2.24E−11 8.3 10.3-D LHG-3.71E+05 0.4 1.21E−05 2.2 3.27E−11 5.7 10.4-D LHG- 3.83E+05 0.4 1.07E−052.4 2.80E−11 6.6 10.5-D LHG- 2.84E+05 0.5 6.15E−06 4.3 2.16E−11 8.610.6-D LHG-10 2.39E+05 1.0 3.12E−05 1.0 1.31E−10 1.0 LHG- 2.87E+05 0.86.38E−06 4.9 2.22E−11 5.9 10.3 LHG- 4.48E+05 0.5 1.30E−05 2.4 2.91E−114.5 10.4 LHG- 4.15E+05 0.6 1.37E−05 2.3 3.31E−11 4.0 10.5 LHG- 2.76E+050.9 4.40E−06 7.1 1.59E−11 8.2 10.6

Example 6: Two Anti-hGARP mAbs (MHGARP8 and LHG-10) Inhibit ActiveTGF-β1 Production by Human Tregs

Stimulated human Tregs produce active TGF-β1 close to their cellsurfaces. Autocrine and paracrine TGF-β1 activity induces SMAD2phosphorylation in Tregs themselves, and in Th cells co-cultured withTregs (Stockis, J. et al. Eur. J. Immunol. 2009, 39:869-882). To test ifGARP is required for TGF-β1 activation by Tregs, human Tregs werestimulated in the presence or absence of anti-hGARP mAbs, andphosphorylation of SMAD2 was measured by Western Blot. As a source ofhuman Tregs we used CD4+CD25^(hi)CD127^(lo) cells sorted from PBMCs andamplified in vitro during 12-14 days (Gauthy E et al PLoS One. 2013 Sep.30; 8(9):e76186). As determined by methyl-specific qPCR, amplified cellpopulations contain 44 to 82% cells with a demethylated FOXP3i1 allele,indicating that they are still highly enriched in Tregs.

As expected, phosphorylated SMAD2 was detected in the stimulated Tregs,but not in non-stimulated Tregs, nor in Tregs stimulated in the presenceof a neutralizing anti-TGF-β1 antibody (FIGS. 11A-11B). PhosphorylatedSMAD2 was greatly reduced in Tregs stimulated in the presence of MHGARP8(named MHG-8 on FIG. 11A) or LHG-10 (FIG. 11B), indicating that thesetwo anti-hGARP mAbs block active TGF-β production. The 29 other newanti-hGARP mAbs, as well as four commercially available anti-hGARP mAbs,did not block TGF-β production by Tregs (FIGS. 11A-11B).

The inhibitory activity of MHGARP8 and LHG-10 shows that GARP isrequired for active TGF-β1 production by human Tregs.

Example 7: MHGARP8 and LHG-10 Inhibit the Suppressive Activity of HumanTregs in Vitro

We previously showed that human Tregs suppress other T cells at least inpart through production of active TGF-β1 (Stockis, J. et al. Eur. J.Immunol. 2009, 39:869-882). We therefore tested whether MHGARP8 (MHG-8)and LHG-10 also inhibit human Treg function in in vitro suppressionassays. A Treg clone was used as a source of Tregs, and freshly isolatedCD4⁺CD25⁻CD127^(hi) cells or a CD4⁺ T cell clone (Th cells) as targetsfor suppression. Tregs and Th cells were stimulated with >CD3 and >CD28in the presence or absence of various additional mAbs. As shown in FIG.12A, clone Treg A1 inhibited the proliferation of CD4⁺CD25⁻CD127^(hi) Thcells by 66% in the absence of anti-hGARP mAb. Suppression was reducedto 36% and 32% in the presence of MHG-8 or LHG-10, respectively, but wasnot reduced in the presence of 6 other anti-hGARP mAbs. Suppression byclone Treg A1 on another Th target (clone Th A2) was also measured inthe presence of MHGARP8, an anti-hTGF-β1 mAb or an isotype control.MHGARP8 (MHG-8) inhibited the in vitro suppressive activity of Treg A1in a manner similar to that of the anti-TGF-β1 antibody, whereas theisotype control showed no effect (FIG. 12B).

Example 8: Epitopes Recognized by Inhibitory Anti-hGARP mAbs

Only a minority (2/35) of anti-hGARP mAbs block active TGF-β productionand suppression by Tregs. This could be due to their ability to bindepitope(s) that are distinct from those bound by non-inhibitory mAbs.Therefore the regions required for binding by inhibitory andnon-inhibitory mAbs were mapped.

GARP associates with pro- or latent TGF-β1 to form disulfide-linkedGARP/TGF-β1 complexes (FIG. 13A and Stockis 2009 Eur. J. Immunol. 2009.39: 3315-3322 and Gauthy E et al). We first sought to determine whetheranti-hGARP mAbs also bind GARP/TGF-β1 complexes, usingco-immunoprecipitation (IP) experiments in murine BW cells transfectedwith hGARP and hTGFB1. Thirty-two anti-hGARP mAbs were tested: 31 mAbsof the instant invention and the commercially available Plato-1 mAb. AllmAbs efficiently immunoprecipitated GARP (top panel of FIG. 14A, showingIPs with 12 representative mAbs). Pro-TGF-β1, as well as LAP and matureTGF-β1 (i.e. latent TGF-β1) were co-immunoprecipitated with 24 mAbsindicating that they bind GARP/TGF-β1 complexes (6 mAbs shown in FIG.14A, middle and lower panels). In contrast, 8 mAbs (3 shown in FIG. 14A)did not co-immunoprecipitate pro- or latent TGF-β1, suggesting they bindfree GARP but not GARP/TGF-β1 complexes.

This was confirmed by FACS analyses of transfected 293T cells (FIG.14B). Untransfected 293T cells express no GARP and very low levels ofendogenous TGF-β1. No latent TGF-β is detected on their surface with ananti-LAP antibody. Transfection of GARP or TGFB1 alone induces no or lowsurface LAP, respectively, whereas co-transfection of GARP and TGFB1induces high surface LAP as a result of latent TGF-β1 binding andpresentation by GARP (FIG. 14B, left histograms). Three groups ofanti-hGARP mAbs emerged from the analysis of transfected 293T cells, andare classified in 3 columns in FIG. 13B. A first group (left column)comprises the 8 mAbs that did not co-immunoprecipitate pro- or latentTGF-β1: they bound 293T cells transfected with hGARP alone, but not withhGARP and hTGFB1. This confirms that these mAbs bind free GARP only, asbinding to surface GARP is lost in the presence of TGF-β1 (FIG. 14B,shows 3 representative mAbs of this group). A second group comprisesmost other mAbs (19 mAbs, middle column of FIG. 13B): they bound 293Tcells equally well upon transfection with hGARP alone or with hGARP andhTGFB1, indicating that they bind both free GARP and GARP/TGF-β1complexes (FIG. 14B, shows 6 mAbs of this group). Interestingly, a thirdgroup of 5 mAbs bound 293T cells transfected with hGARP and hTGFB1, butnot cells transfected with hGARP alone (right column of FIG. 13B). ThesemAbs bind GARP/TGF-β1 complexes but not free GARP, and includeinhibitory MHGARP8 (MHG-8) and LHG-10 (FIG. 14B, shows 3 mAbs of thisgroup).

From the above, we concluded that most mAbs bind free GARP only (8/32)or free GARP and GARP/TF-β1 complexes (19/32). Only five mAbs, includinginhibitory MHGARP8 (MHG-8) and LHG-10 but also three non-inhibitorymAbs, bind GARP/TGF-β1 complexes, but not free GARP. This pattern ofrecognition does not explain why only MHGARP8 and LHG-10 are inhibitory.

We next sought to define the regions of hGARP required for binding bythe various mAbs. The vast majority of the anti-hGARP mAbs do notcross-react on mouse GARP (mGARP). Therefore plasmids were constructedencoding HA-tagged mGARP/hGARP chimeras (FIG. 15A, left panel) andtransfected into 293T cells, with or without hTFGB1 depending on thebinding requirements determined above. All chimeras were expressed atsimilar levels on the surface of 293T cells, as evidenced by stainingwith an anti-HA mAb (FIG. 15A, histograms on the right). Bindingpatterns to mGARP/hGARP chimeras (FIG. 15A, 10 representative mAbs)allowed to identify the region of hGARP required for binding by eachanti-hGARP mAb. This is summarized in FIG. 15B, where mAbs aredistributed in rows corresponding to various regions of hGARP: mAbs inthe first row require a region comprising amino-acids 20 to 101(hGARP₂₀₋₁₀₁), mAbs in the second row require hGARP₁₀₁₋₁₄₁, those in thethird require hGARP₁₄₁₋₂₀₇, the fourth, hGARP₂₆₅₋₃₃₂, and finally, afifth group requires hGARP₃₃₂₋₆₂₈. However, even when considering theregions required for binding, the epitope recognized by inhibitoryMHGARP8 (named MHG-8 on the figure) and LHG-10 could not bedistinguished from that of non-inhibitory mAbs: MHGARP8 and LHG-10, likeLHG-3, -12 and -13, bind GARP/TGF-β complexes that contain hGARP₁₀₁₋₁₄₁.

Sequences of mouse and human GARP₁₀₁₋₁₄₁ differ at 14 amino-acid (aa)positions, comprising three clusters of three contiguous positions (FIG.15B, left panel). We constructed three mutated versions of hGARP. Ineach mutant, a series of three contiguous aa from region 101-141 werereplaced by the aa found in mGARP. We transfected 293T cells with theHA-tagged mutants, alone or with hTGFB1 depending on the bindingrequirement of the mAbs tested. Binding patterns to mutants revealedthree types of mAbs (FIG. 15B, right panel), which required amino-acidshGARP₁₁₁₋₁₁₃, hGARP₁₂₆₋₁₂₇, or hGARP₁₃₇₋₁₃₉ for binding, respectively.Six mAbs, including MHGARP8 (named MHG-8 on the figure) and LHG-10,required hGARP₁₃₇₋₁₃₉ (FIG. 13B). Whereas four of six can bind freehGARP, MHG-8 and LHG-10 are the only mAbs that require hGARP₁₃₇₋₁₃₉ inthe context of GARP/TGF-β1 complexes.

From the above, we concluded that inhibition of TGF-β production byMHGARP8 and LHG-10 is associated with the ability to bind an epitopethat is distinct from those recognized by all other, non-inhibitory,anti-hGARP mAbs.

Example 9: Inhibition of Human Tregs Function by Anti-hGARP In Vivo

We next sought to evaluate whether inhibitory anti-hGARP mAbs couldinhibit human Treg function in vivo. We used a model of xenogeneicgraft-versus-host disease (GVHD) induced by transfer of human peripheralblood mononuclear cells (PBMCs) into immuno-compromisedNOD/Scid/IL2Rg-(NSG) mice. NSG mice have defective cytokine signalingand lack functional T, B and NK cells, allowing very efficientengraftment of human T cells upon i.v. injection of PBMCs. Thirty toforty days after PBMC transfer, recipient mice develop xenogeneic GVHD,due to the activity of human cytotoxic T lymphocytes against murinetissues (Shultz, Nat Rev Immunol. 2012 November; 12(11):786-98). In thismodel, co-transfer of human Tregs with human PBMCs attenuates GVHD(Hannon et al. Transfusion. 2014 February; 54(2):353-63), providing amodel to test the inhibitory activity of anti-hGARP mAbs on human Tregsin vivo.

We transferred human PBMCs (3×10⁶/mouse) with or without autologousTregs (1.5×10⁶/mouse) in NSG mice (FIG. 16A). As a source of humanTregs, we used blood CD4⁺CD25^(hi)CD127^(lo) cells that had been shortlyamplified in vitro, as described above. In addition, mice were injectedwith MHGARP8 (named MHG-8 on the figure), anti-TGF-β1, an isotypecontrol or PBS, one day before the graft and weekly thereafter.Objective signs of GVHD were monitored bi-weekly, to establish a diseasescore based on weight loss, reduced mobility, anemia or icterus, andhair loss. We performed four independent experiments (FIG. 16B), anddetailed results are shown for one (FIG. 16C). Depending on theexperiment, onset of disease (mean GVHD score >1) was observed 28 to 41days after PBMC transfer in groups of mice that received no mAb or anisotype control. Co-transfer of Tregs delayed disease, which occurred 46to 72 days after transfer, indicating that human Tregs were able tosuppress human T cell responses against xenogeneic antigens.Administration of MHGARP8 to mice transferred with PBMCs and Tregsabrogated the protective effect of Tregs: disease occurred as early asin mice receiving PBMCs only (28 to 44 days after transfer). Inhibitionof Treg suppressive function by MHGARP8 was similar to that observedwith a neutralizing anti-TGF-β1 antibody. An isotype control had noeffect.

Altogether, this shows that MHGARP8 inhibits the immune-suppressivefunction of human Tregs in vivo.

We verified that MHGARP-8 did not aggravate GVHD in mice grafted withPBMCs alone, thus that its effect depended on the co-injection of Tregs(FIG. 17). We also examined whether abrogation of Treg protection byMHGARP-8 depended on its ability to block TGF-β production. MHGARP-8 wascompared to LHG-10.6, which also blocks TGF-β production by Tregs, andto LHG-3, which does not. Antibody LHG-10.6 is a variant of LHG-10 withincreased affinity for GARP/TGF-β1 complexes that was selected by phagedisplay from Fabs in which the heavy chain of LHG-10 was combined to theVK library. Like MHGARP-8, LHG-10.6 aggravated GVHD, whereasnon-blocking antibody LHG-3 had no effect (FIG. 17). This suggested thatMHGARP-8 and LHG-10.6 abrogate Treg protection by blocking Tregproduction of TGF-β1, and not by inducing Treg depletion. To furtherexclude the latter possibility, a mutated version of LHG-10.6, namedLHG-10.6N297Q, was also tested. The N297Q mutation results in loss of Fcglycosylation, thus loss of Fc receptor- and C1q-binding, andconsequently loss of ADCC and CDC functions. LHG-10.6N297Q aggravatedGVHD in mice grafted with PBMCs and Tregs as potently as LHG-10.6,confirming that anti-GARP antibodies do not act by depleting Tregs (FIG.17).

Human cytokines in the serum of mice 20 days after cell transfer weremeasured (FIG. 18A). Human IL-2 and IFNγ were detected at high levels inmice grafted with PBMCs only, indicating a strong xenogeneic activationof human T cells. They were significantly reduced by the cotransfer ofTregs, confirming suppressive activity. MHGARP-8 decreased thesuppression by Tregs, but had no effect in mice transferred with PBMCsalone. Finally, IL-10 levels were not increased but instead reduced inthe presence of Tregs, suggesting that Tregs do not suppress throughproduction of IL-10 in this model (FIG. 18A). Noteworthy, effects ofMHGARP-8 on suppression of cytokine production were measured at an earlytime point before disease onset, to preclude confounding effects fromsevere inflammation that develops in some groups at later time points.This likely explains why the effects of MHGARP-8 are less pronounced oncytokine production than on disease progression.

In spleens collected 20 days after transfer, human hematopoietic cells(hCD45+) comprised mostly T lymphocytes (CD4+ and CD8+), which hadconsiderably proliferated in mice grafted with PBMCs alone. Thisproliferation was inhibited by the co-transfer of Tregs, an effect thatwas decreased by MHGARP-8 (FIG. 18B). Notably, the numbers andproportions of Tregs (hCD3+hCD4+hFOXP3+ cells or cells with ademethylated FOXP3i1 allele) were not reduced in mice treated withMHGARP-8. On the contrary, Treg numbers were significantly increased inmice transferred with Tregs and treated with MHGARP-8 as compared tountreated mice (FIG. 18C). This suggests that the MHGARP-8-mediatedblockade of autocrine TGF-β1 activity favors Treg proliferation, whileconcomitantly inhibiting Treg function. It also supports the hypothesisthat inhibitory anti-GARP mAbs do not act by depleting Tregs.

However, it could still be that inhibitory mAbs deplete a minorsubpopulation of Tregs without affecting total Treg numbers. Forexample, this could occur if only a small proportion of Tregs expressedGARP as a result of activation in this model. In vitro, GARP was shownto be expressed only on activated Tregs. First, the proportions andnumbers of GARP+Tregs were measured at several time points after thetransfer of human PBMCs±Tregs in NSG mice (FIG. 18D). Three days aftertransfer, approximately 50% of CD4+FOXP3+ cells expressed GARP,indicating that GARP+ cells do not represent a minor subpopulation ofTregs. Proportions of GARP+ cells decreased progressively to 10-20% ofCD4+FOXP3+ by day 20, and no difference was observed between untreatedmice and mice treated with the anti-GARP mAb LHG-10.6, which isinhibitory, or LHG-3, which is not. The numbers of GARP+Tregs were notreduced in mice treated with either anti-GARP mAb by comparison tountreated mice. They were even increased at day 20 in mice treated withLHG-10.6 compared to all other conditions. Second, anotheridentification procedure for activated human Tregs, as proposed byMiyara et al. was used (M. Miyara, et al. Immunity 30, 899-911 (2009))who defined these cells as a subset of FOXP3+Treg cells characterized byhigh FOXP3 levels and no CD45RA expression (CD4+FOXP3hiCD45RA− cells). Asubpopulation of human FOXP3hi cells appeared in mice 7 days after thetransfer of human PBMCs±Tregs. FOXP3hi cells expressed the highestlevels of GARP and most were CD45RA−. Therefore in vivo, GARP expressionwas maximal in the activated human CD4+FOXP3hiCD45RA− Tregs. The numbersof CD4+FOXP3hiCD45RA− cells were not decreased in mice treated withanti-GARP mAbs by comparison to untreated mice (FIG. 18D), confirmingthat inhibitory anti-GARP mAbs did not act in this model by depletingGARP-expressing cells.

Altogether, these results indicate that the inhibitory anti-GARP mAbsare capable of inhibiting the immunosuppressive activity of human Tregsin vivo without inducing Treg depletion.

Example 10: X-Ray Crystal Structures of the Fab-Fragment Generated fromAntibody MHGARP8 in Complex with the GARP/TGF-β Complex

The Fab fragment of MHG-8 was prepared by papain digestion of MHG-8 andpurified using Protein A affinity chromatography and gel filtrationchromatography. The MHG-8 Fab fragment was added to the GARP/TGFβcomplex to allow binding of the Fab to its antigen. The Fab-fragmentpurified in this way was mixed with the GARP/TGFbeta complex and appliedon a gel filtration column in 20 mM Tris/HCl pH 8.0, 50 mM NaCl. TheFab/GARP/TGFβ complex was concentrated on a 50 kD Vivascienceultrafiltration device to a final concentration of 18 mg/mL, asdetermined by Nanodrop (UV). This MHG-8 Fab/GARP/TGFβ complex (whereTGFβ is comprised of LAP and mature TGFβ) was purified and used forcrystallization.

Methods

Crystallisation

The purified protein was used in crystallisation trials employing astandard screen with approximately 1,200 different conditions.Conditions initially obtained have been optimised using standardstrategies, systematically varying parameters critically influencingcrystallisation, such as temperature, protein concentration, drop ratio,and others. These conditions were also refined by systematically varyingpH or precipitant concentrations.

Data Collection and Processing (Table 5)

The application of the Free Mounting System (FMS) was necessary toobtain well diffracting crystals. The crystals were coated with oil andtransferred to the N2 cryo-stream at 100K. Crystals have beenflash-frozen and measured at a temperature of 100 K. The X-raydiffraction data have been collected from complex crystals at the SWISSLIGHT SOURCE (SLS, Villigen, Switzerland) using cryogenic conditions.The crystals belong to space group P 21. Data were processed using theprograms XDS and XSCALE.

TABLE 5 X-ray source PXI/X06SA (SLS¹) Wavelength [Å] 1.00001 DetectorPILATUS 6M Temperature [K] 100 Space group P 2₁ Cell: a; b; c; [Å]103.89; 175.11; 145.81 α; β; γ; [°] 90.0; 92.2; 90.0 Resolution [Å] 3.15(3.40-3.15) Unique reflections 83391 (16806) Multiplicity 3.0 (2.9)Completeness [%] 92.7 (92.2) R_(sym) [%]³ 8.9 (67.7) R_(meas) [%]⁴ 10.7(81.7) Mean(I)/sd⁵ 10.24 (1.75) ¹SWISS LIGHT SOURCE (SLS, Villigen,Switzerland) ²values in parenthesis refer to the highest resolution bin.${\,^{3}{Rsym}} = {{\frac{\sum\limits_{h}^{\;}{\text{?}{{{\hat{I}}_{h} - I_{h,i}}}}}{\sum\limits_{h}^{\;}{\text{?}I_{h,i}}}\mspace{14mu} {with}\mspace{14mu} {\hat{I}}_{h}} = {\frac{1}{n_{h}}\text{?}I_{h,i}}}$where I_(h,i) is the intensity value of the ith measurement of h${\,^{4}{Rmeas}} = {{\frac{\sum\limits_{h}^{\;}{\text{?}{{{\hat{I}}_{h} - I_{h,i}}}}}{\sum\limits_{h}^{\;}{\text{?}I_{h,i}}}\mspace{14mu} {with}\mspace{14mu} {\hat{I}}_{h}} = {\frac{1}{n_{h}}\text{?}I_{h,i}}}$where I_(h,i) is the intensity value of the ith measurement of h⁵calculated from independent reflections?indicates text missing or illegible when filed

Structure Modelling and Refinement

The phase information necessary to determine and analyse the structurewas obtained by molecular replacement. The published structures oflatent TGFbeta (PDB-ID 3RJR), Leucine-rich repeat andimmunoglobulin-like domain-containing nogo receptor interacting protein1 (PDB-ID 4OQT) and Fab-fragment (PDB-ID 1FNS) were used as a searchmodels. Subsequent model building and refinement was performed accordingto standard protocols with the software packages CCP4 and COOT. For thecalculation of the free R-factor, a measure to crossvalidate thecorrectness of the final model, about 0.6% of measured reflections wereexcluded from the refinement procedure (see Table 6). Automaticallygenerated local NCS restraints have been applied (keyword “ncsr local”of newer REFMAC5 versions). The water model was built with the “Findwaters”-algorithm of COOT by putting water molecules in peaks of theFo-Fc map contoured at 3.0 followed by refinement with REFMAC5 andchecking all waters with the validation tool of COOT. The criteria forthe list of suspicious waters were: B-factor greater 80 A2, 2Fo-Fc mapless than 1.2 σ, distance to closest contact less than 2.3 Å or morethan 3.5 Å. The suspicious water molecules and those in the ligandbinding site (distance to ligand less than 10 Å) were checked manually.

The occupancy of side chains, which were in negative peaks in the Fo-Fcmap (contoured at −3.0 σ), were set to zero and subsequently to 0.5 if apositive peak occurred after the next refinement cycle. The RamachandranPlot of the final model shows 79.3% of all residues in the most favouredregion, 19.4% in the additionally allowed region, and 1.1% in thegenerously allowed region. The residues Asn31(L), Ala51(L), Asn31(κ),and Ala51(κ) are found in the disallowed region of the Ramachandran plot(Table 5). They are either confirmed by the electron density map orcould not be modelled in another sensible conformation. Statistics ofthe final structure and the refinement process are listed in Table 6.

TABLE 6 Resolution [Å] 145.70-3.15 Number of reflections (working/test)82901/490 R_(cryst) [%] 24.1 R_(free) [%]² 27.5 Total number of atoms:Protein 25251 Water 0 β-D-mannose 44 β-D-N-acetyl-glucose 168 Deviationfrom ideal geometry:³ Bond lengths [Å] 0.007 Bond angles [°] 1.26 BondedB's [Å²]⁴ 12.5 Ramachandran plot:⁵ Most favoured regions [%] 79.3Additional allowed regions [%] 19.4 Generously allowed regions [%] 1.1Disallowed regions [%] 0.1 ¹values as defined in REFMAC5, without sigmacut-off ²Test-set contains 0.6% of measured reflections ³Root meansquare deviations from geometric target values ⁴Calculated with MOLEMAN⁵Calculated with PROCHECK

Results

The structure of the Fab:GARP/TGF-β complex was solved at a resolutionof 3.15 Å.

The crystal structure of the Fab:GARP/TGF-β complex allowed theidentification of the epitope recognized by the antibody MHGARP8. TheFab-fragment binds a composite three dimensional epitope on theFab:GARP/TGF-β complex. The interaction surface on the Fab:GARP/TGF-βcomplex is formed by several continuous and discontinuous sequences fromboth hGARP (Tables 7a and 7b respectively) and TGF-β (Table 7c), showinginteractions between TGFβ residues and MHG-8 heavy chain residues.

TABLE 7a Interactions between GARP residues (left side) and MHG-8 heavychain residues (right side) Residue Number Chain Residue Number ChainTyr 137 hGARP Asp 103 Heavy chain Ser 138 hGARP Tyr 102 Heavy chain Gly139 hGARP Tyr 102 Heavy chain Asp 103 Heavy chain Tyr 104 Heavy chainLeu 140 hGARP Tyr 104 Heavy chain Glu 142 hGARP Tyr 102 Heavy chain Arg143 hGARP Tyr 104 Heavy chain Asp 105 Heavy chain Thr 162 hGARP Asp 103Heavy chain Arg 163 hGARP Asn 100 Heavy chain Tyr 101 Heavy chain Tyr102 Heavy chain Thr 165 hGARP Tyr 101 Heavy chain Tyr 102 Heavy chainArg 166 hGARP Tyr 101 Heavy chain His 167 hGARP Tyr 101 Heavy chain Tyr102 Heavy chain Glu 189 hGARP Tyr 101 Heavy chain

TABLE 7b Interactions between GARP residues (left side) and MHG-8 lightchain residues (right side) Residue Number Chain Residue Number ChainThr 113 hGARP Trp 92 Light chain Ser 93 Light chain Ala 114 hGARP Ser 93Light chain Ser 116 hGARP Trp 92 Light chain Ala 117 hGARP His 28 Lightchain Ile 29 Light chain Trp 92 Light chain Gly 118 hGARP His 28 Lightchain Lys 30 Light chain Trp 92 Light chain Gly 119 hGARP Trp 92 Lightchain Gly 139 hGARP Trp 32 Light chain Glu 142 hGARP Lys 30 Light chainTrp 32 Light chain Arg 143 hGARP Trp 32 Light chain Tyr 91 Light chainTrp 92 Light chain Ser 93 Light chain Trp 96 Light chain Leu 144 hGARPTrp 92 Light chain Leu 145 hGARP Lys 30 Light chain Gly 146 hGARP Lys 30Light chain Arg 170 hGARP ASN 31 Light chain Trp 32 Light chain

TABLE 7c Interactions between TGFβ residues (left side) and MHG-8 heavychain residues (right side) Residue Number Chain Residue Number ChainArg 58 lTGFbeta Asp 54 Heavy chain Glu 100 lTGFbeta Asn 73 Heavy chainGlu 146 lTGFbeta Arg 68 Heavy chain Gln 269 lTGFbeta Asp 54 Heavy chainGly 55 Heavy chain Ser 56 Heavy chain Thr 57 Heavy chain His 270lTGFbeta Thr 57 Heavy chain Tyr 59 Heavy chain Arg 68 Heavy chain Ile 69Heavy chain Leu 271 lTGFbeta Thr 57 Heavy chain Tyr 59 Heavy chain Gln272 lTGFbeta Thr 57 Heavy chain Asp 58 Heavy chain Tyr 59 Heavy chainSer 273 lTGFbeta Thr 57 Heavy chain Tyr 284 lTGFbeta Gly 26 Heavy chainPhe 27 Heavy chain Ser 28 Heavy chain Ser 76 Heavy chain Tyr 336lTGFbeta Tyr 104 Heavy chain Ser 337 lTGFbeta Asp 54 Heavy chain Lys 338lTGFbeta Trp 52 Heavy chain Asp 54 Heavy chain Ser 56 Heavy chain Tyr104 Heavy chain Ala 341 lTGFbeta Asp 54 Heavy chain Gln 345 lTGFbeta Thr30 Heavy chain

Example 11: Impact of Mutations in GARP or TGF-β1 on the Binding andActivity of Inhibitory Anti-GARP Antibodies MHG-8 and LHG-10

Impact of Mutations in GARP or TGF-β1 on the Binding of InhibitoryAnti-GARP Antibodies MHG-8 and LHG-10 Used in Saturating Conditions.

Resolution of the crystal structure of MHG-8 Fab/GARP/TGF-β1 complexesand analysis with the CONTACT software allowed the identification of 22amino-acids in GARP, 8 amino-acids in LAP and 6 amino-acids in matureTGF-β1 that are located less than 5 Å away from an amino-acid of thelight or heavy chain of the MHG-8 Fab (Tables 7a, b and c, and Table 8).Directed mutagenesis was used to construct plasmids encoding HA-taggedforms of GARP or TGF-β1 that are mutated at one, two, or three of these36 positions (all mutations constructed are indicated in Table 8). 293Tcells were then transfected with these plasmids and the ability ofanti-GARP, anti-LAP or anti-HA antibodies to bind to the GARP/TGF-β1complexes containing mutated forms of GARP or TGF-β1 was determined byflow cytometry. Antibodies were used at a saturating concentration of 5μg/ml for labeling. Binding of each antibody to a given mutantGARP/TGF-β1 complex was compared to their binding to wild type (WT)complexes, by determining an I_(m/wt) value (i.e. intensity of bindingto mutant by comparison to WT). This value was calculated as follows:I_(m/wt) of antibody X=[Geom on mutant−Geom on control]/[Geom on WT−Geomon control] where Geom corresponds to the geometric mean of thefluorescence intensity measured by FACS on 293T cells transfected withempty plasmid (control) or plasmids encoding the mutant or WT forms ofGARP/TGF-β1 complexes.

The level of residual binding of anti-GARP or anti-LAP antibodies toeach mutant was then calculated by comparison to WT, taking into accountthe expression levels of mutant and WT forms as measured with anti-HAantibodies. Residual binding was calculated as follows: Residual bindingby an anti-GARP or anti-LAP antibody=[I_(m/wt) with anti-GARP oranti-LAP antibody]/[I_(m/wt) with anti-HA antibody] Results obtained onall mutants are detailed in FIGS. 19A-19D for binding by inhibitoryanti-GARP antibodies MHG-8 and LHG-10, non-inhibitory anti-GARP antibodyMHG-6, and anti-LAP antibody, respectively. They are also summarized inTable 8 for MHG-8 and LHG-10.

TABLE 8 Effects of GARP and TGFβ1 mutations on the binding andinhibitory activity of mAbs MHG-8 and LHG-10 CDR of Effects of Effectsof MHG-8 Aa of GARP, Corresponding mutations on mutations on in contactLAP or mature aa in mouse MHG-8 binding LHG-10 binding with TGF-β1 inGARP/TGF-β1 and activity and activity GARP, contact complexes, Loss ofLoss of LAP or with Fab Mutation if different Loss of Reduced inhibitoryLoss of Reduced inhibitory mature MHG-8¹ constructed from human binding²avidity³ Activity⁴ binding² avidity³ activity⁴ TGF-β1 GARP Thr113 M111T/Met − − − − − − L3 A112G/ T113M Ala114 A114R − nt − − nt − L3 Ser116S116N Asn − nt − − nt − L3 Ala117 A117R − nt − − nt − L1/L3 Gly118G118L/ − − + − − ? L1/L3 Gly119 G119L L3 Tyr137 Y137H His − − + +/− − −H3 Ser138 S138G Gly − − − − − − H3 Gly139 G139N Asn + NE + − − − H3/ L1Y137H/ + NE + + + + S138G/ G139N Leu140 L140K/ + NE + − − − H3 Glu142E142L H3/L1 Arg143 R143A + NE nt − nt nt H3/ R143Y + NE + +/− − ? L1/ L3Leu144 L144Q/ Mutant not evaluable because not L3 Leu145 L145Q/expressed on surface L1 Gly146 G146K L1 Thr162 T162D − + ? + + + H3Arg163 R163E + + + + + + H3 R163A/ + nt nt − nt nt T165A R163E/ + NE nt+/− nt nt T165A Thr165 T165A Ala − − − − − − H3 Arg166 R166M/ − + − − −− H3 His167 H167E H3 Arg170 R170A Trp − − − − − − L1 Glu189 E189A − − −− − − H3 LAP Arg58 R58A − − − − − + H2 Glu100 E100A − nt − − nt − H2Glu146 E146R − − − − − − H2 Gln269 Q269Y − − − − − − H2 His270 H270Y − −− − − − H2 Leu271 H2 Gln272 Q272Y His − − − − − − H2 Ser273 H2 L271R/ −nt − − nt − Q272Y/ S273W Mature TGF-β1 Tyr284 Y284A − − − − − − H1Tyr336 Y336A − + − +/− − ? H3 Y336Q − nt nt − − nt Ser337 S337A − nt − −− − H2 Lys338 K338E + + + + − + H2/ H3 Ala341 A341Y − nt − − nt − H2Gln345 Q345A − nt − − nt − H1 ¹Amino-acids located at less than 5angstrom from an amino-acid of MHG-8 as determined with the CONTACTsoftware ²+: <50% residual binding on mutant by comparison to WT; −:≥50% residual binding on mutant by comparison to WT; +/−: residualbinding ≥50% but error bar (sdt deviation) crosses the 50% threshold ³+:ratio of EC50 (mutant vs WT) >2; −: ratio of EC50 (mutant vs WT) ≤2; NE:non evaluable; nt: not tested ⁴+: residual inhibitory activity <21%;not: not tested; ?: not conclusive, should be re-tested

FIG. 19A, demonstrates that binding by MHG-8 was lost (i.e. residualbinding <50%) on the following GARP mutants:

-   -   triple mutation Y137H/S138G/G139N (2% residual binding),        confirming previous observations [Cuende et al., 2015];    -   single mutation G139N (2% residual binding), but not single        mutations Y137H and S138G, indicating a more prominent role of        GARP Gly139 than Tyr137 and Ser138 in binding by MHG-8;    -   double mutation L140K/E142L (7% residual binding);    -   single mutations R143A and R143Y (1% residual binding);    -   double mutations R163A/T165A (36% residual binding) and        R163E/T165A (4% residual binding);    -   single mutation R163E (19% residual binding), but not single        mutation T165A, indicating a more prominent role of Arg163 than        Thr165 for binding by MHG-8.

Altogether, of the 22 aa in GARP that are in contact with the MHG-8 Fabas determined by analysis of the crystal structure, only 5 aa (Gly139,Leu140/Glu142, Arg143 and Arg163) appear to be required for binding tothe MHG-8 mAb.

In addition, FIG. 19A demonstrates that only one mutation in TGF-β1results in loss of binding by MHG-8. This mutation corresponds to K338E(25% residual binding). Thus, of the 8 aa from LAP and 6 aa from matureTGF-β1 that are in contact with MHG-8 Fab in the crystal, only one aa inmature TGF-β1 (Lys338) is required for binding to the MHG-8 mAb.

FIG. 19B shows residual binding by LHG-10 on GARP mutants. The resultscan be summarized as follows:

-   -   triple mutation Y137H/S138G/G139N induces loss of binding by        LHG-10 (6% residual binding), confirming previous observations        [Cuende et al., 2015];    -   single mutation Y137H induces a partial loss of binding (62%        residual binding, but standard deviation of the mean value        crosses the 50% threshold), whereas single mutations G139N and        S138G do not affect binding. The mode of binding of LHG-10 to        GARP is thus different from that of MHG-8 (see above);    -   double mutation L140K/E142L does not induce loss of binding to        LHG-10, in contrast to what was observed for MHG-8, confirming        that the mode of binding of LHG-10 to GARP is different from        that of MHG-8;    -   single mutation R143Y only induces a partial loss of binding        (68% residual binding), whereas mutation R143A does not. Thus,        aa Arg143 appears more important for binding by MHG-8 than        binding by LHG-10;    -   single mutation T162D induces loss of binding by LHG-10 (49%        residual binding), whereas it had no effect on binding by MHG-8;    -   single mutation R163E induces loss of binding by LHG-10 (48%        residual binding).

Altogether, of the 22 aa in GARP that are in contact with the MHG-8 Fabas determined by analysis of the crystal structure, only two aa (Thr162and Arg163) appear to be required for binding by the LHG-10 mAb. Inaddition, two other aa (Tyr137 and Arg143) appear to also play a role,as mutations of the aa induce partial loss of binding by LHG-10. Of thefour aa important for binding by LHG-10, two are also required forbinding by MHG-8 (Arg143 and Arg163), whereas two are unique to LHG-10(Tyr137 and Thr 162).

FIG. 19B, also shows that one mutation in mature TGF-β1 (K338E, 15%residual binding) induces loss of binding to LHG-10. This mutation alsoinduced loss of binding to MHG-8. One other mutation in mature TGF-β1(Y336A, 56% residual binding) induced partial loss of binding to LHG-10,whereas it did not affect binding to MHG-8.

As expected, none of the mutations tested induced loss of binding toMHG-6, a non-inhibitory anti-GARP antibody that binds GARP/TGF-β1complexes on an epitope distant from that bound by MHG-8 and LHG-10(FIG. 19C). Similarly, none of the mutations induced loss of binding tothe anti-LAP antibody (FIG. 19D).

Impact of Mutations in GARP or TGF-β1 on the Avidity of InhibitoryAnti-GARP Antibodies MHG-8 and LHG-10 for GARP/TGF-β1 Complexes.

Mutations that do not induce loss of binding by MHG-8 or LHG-10 (>50%residual binding) could nevertheless induce a reduced avidity of theantibodies for the mutated GARP/TGF-β1 complexes by comparison to WT.The avidity of each antibody on the WT and on the various mutated formsof GARP/TGF-β1 complexes was determined by performing FACS analyses of293T cells transfected as described above, using 5-to-5 serial dilutionsof antibodies for labeling (final concentrations of 5, 1, 0.2, 0.04,0.008 and 0.0016 μg/ml). Intensities of staining at each concentrationof antibody relative to the maximal intensity measured at 5 μg/ml weredetermined as follows:

Relative staining intensity=[Geom with X μg/ml of antibody−Geom in theabsence of antibody]/[Geom with 5 μg/ml of antibody−Geom in the absenceof antibody]

The relative staining intensities were plotted according to theconcentration of antibody used for staining, and a non-linear regressionanalysis was used with the Prism software to determine an EC₅₀ value forbinding of the antibody to the WT and to each of the various mutantGARP/TGF-β1 complexes. If the ratio between the EC₅₀ measured on themutant and the EC₅₀ measured on the WT was >2, the mutation wasconsidered to induce a reduced avidity for binding by the antibody. Theratio of EC₅₀(mutant vs WT) for MHG-8 and LHG-10 are indicated beloweach mutation on FIGS. 19A and 19B, respectively. The data from thisexperiment is also summarized in Table 8.

The avidity of the antibodies for mutations that induce a complete lossof binding (<10% residual binding) cannot be evaluated (NE=non evaluablein FIGS. 19A and 19B).

Further, most mutations that induce incomplete loss of binding (10 to50% residual binding) also induce reduced avidity. This is the case formutations R163E in GARP and K338E in TGF-β1 for binding by MHG-8, andfor mutations T162D and R163E in GARP for binding by LHG-10.Unexpectedly, a few mutations that induced partial loss of binding at 5μg/ml did not appear to induce reduced avidity. This was the case formutations Y137H and R143Y in GARP and mutations Y336A and K338E inTGF-β1 for binding by LHG-10. However, in these apparently paradoxicalcases, the concentration of 5 μg/ml of LHG-10 used by default as themaximum concentration did not yield a saturated signal. This couldinduce an error in the calculated EC₅₀, explaining the apparentdiscrepancy.

Interestingly, three mutations that did not induce loss of binding (>50%residual binding) when MHG-8 was used at 5 μg/ml, nevertheless reducedthe avidity of MHG-8 for the GARP/TGF-β1 complexes. These mutationscorrespond to T162D and R166/H167E in GARP, and Y336A in TGF-β1.Mutations T162D in GARP and Y336A in TGF-β1 induced loss of binding byLHG-10, indicating that Thr162 of GARP and Tyr336 of TGF-β1 areimportant amino acids for the binding of both MHG-8 and LHG-10, althoughthe effect of mutations at these positions are not completely identical.

Impact of Mutations in GARP or TGF-β1 on the Inhibitory Activity ofAnti-GARP Antibodies MHG-8 and LHG-10.

Mutations that neither induce loss of binding nor reduce the avidity ofMHG-8 or LHG-10 for GARP/TGF-β1 complexes could nevertheless result in adecreased ability of the antibodies to inhibit active TGF-β1 productionfrom GARP/TGF-β1 complexes. This would indicate the existence of pointsof contact between GARP/TGF-β1 complexes and MHG-8 and/or LHG-10 thatare important for the inhibitory activity of the antibodies but are notrequired for their binding. In order to examine this possibility,functional assays were developed to measure active TGF-β1 productionfrom wild type or mutant GARP/TGF-β1 complexes in transfected 293Tcells, in the presence or absence of anti-GARP antibodies.

To measure the capacity of MHG-8 and LHG-10 to inhibit active TGF-β1production from GARP/TGF-β1 complexes containing mutant forms of GARP, aclone of 293T cells stably transfected with integrin 16, which pairswith the endogenously expressed integrin uV to form a well-knownactivator of latent TGF-β1, was used. This clone, named 293T+ITGB6, wastransiently co-transfected with mutant forms of GARP, with wild typeTGF-β1 and with a CAGA-Luc reporter plasmid in which luciferaseexpression is driven by a TGF-β1 responsive promoter.

To measure the capacity of MHG-8 and LHG-10 to inhibit active TGF-β1production from GARP/TGF-β1 complexes containing mutant forms of TGF-β1,a clone of 293T cells stably transfected with integrin 16 and GARP wasused. This clone, named 293T+ITGB6+GARP, was transiently co-transfectedwith mutant forms of TGF-β1 and with the CAGA-Luc plasmid.

Transiently transfected cells (293T+ITGB6 or 293T+ITGB6+GARP) wereincubated during 24 hours with inhibitory anti-GARP antibodies MHG-8 orLHG-10, or with control antibodies corresponding to a neutralizinganti-TGF-β1 antibody (positive control), or the non-inhibitory anti-GARPantibody LHG-14 (negative control). All antibodies were used at 20μg/ml. Cells were then lysed and incubated with a luciferase substrateand the luminescent signal was measured in a luminometer.

To compare various mutants tested in different experiments, theinhibitory activity of antibodies were experessed as follows:

Residual inhibitory activity (%)=100×[Inhibition by antibody X on themutant complex]/[Inhibition by antibody X on the WT complex]

where Inhibition by antibody X=1 −[luminescent signal with antibodyX]/[luminescent signal with the control, non inhibitory anti-GARPantibody LHG-14]

Results obtained are detailed in FIGS. 20A-20C for MHG-8, LHG-10 andanti-TGF-β1, respectively, and summarized in Table 8.

As expected, mutations that induce loss of binding by MHG-8 or LHG-10(underlined in FIG. 20A and FIG. 20B) induce a complete or almostcomplete loss of their inhibitory activity (residual inhibitory activity<21%). Mutations that reduce the avidity of the antibodies withoutinducing complete loss of binding (indicated in italics in FIG. 20A andFIG. 20B) do not appear to affect the inhibitory activity of theantibodies when used at 20 μg/ml in this assay.

Interestingly, a few mutations that did not induce loss of binding norreduced the avidity of the antibodies induced a severe loss ofinhibitory activity. These mutations are highlighted by a box in FIG.20A and FIG. 20B. They comprise mutations G118L/G119L and Y137H of GARP,on which MHG-8 only exerts 11% and 20% residual inhibitory activity,respectively. These mutations also appear to at least partially affectthe inhibitory activity of LHG-10. Further, mutation R58A does notaffect binding by antibody LHG-10, but completely abolishes itsinhibitory activity. As expected, none of the mutations tested affectthe inhibitory activity of the anti-TGF-β1 antibody, used here as apositive control.

Materials and Methods

Mice

Mice (DBA/2, Balb/c, and NOD.Cg-Prkdcscid Il12rgtmlWjl/SzJ or NSG fromThe Jacskon Laboratory) were bred at the animal facility of theUniversite Catholique de Louvain, Belgium. Handling of mice andexperimental procedures were conducted in accordance with national andinstitutional guidelines for animal care.

Cells and Transfections

P1.HTR cells, a highly transfectable variant of the P815 mastocytomaderived from DBA/2 mice, were electroporated with a plasmid encoding thefull-length human GARP and selected in puromycin (1.6 μg/ml) underlimiting dilution conditions. Two clones expressing high surface hGARP(P1.HTR+hGARP) were isolated and used to immunize H-2d mice. A stableclone of murine BW5147.C2 cells expressing high levels of human GARP(BW5147+hGARP) was derived as described (E. Gauthy et al., PLoS One 8,e76186 (2013)). This clone was electroporated with a plasmid encodingfull-length human TGF-b1, and selected in neomycin (3 mg/ml) underlimiting dilution conditions. A subclone expressing high levels ofsurface hGARP/hTGF-β1 complexes (BW5147+hGARP+hTGFB1) was isolated andused to immunize llamas. Human Treg and Th clones were derived andcultured as previously described (J. Stockis, et al. Eur. J. Immunol.39, 869-882 (2009).). Total human PBMCs were purified from the blood ofhemochromatosis donors by centrifugation on a Lymphoprep® gradient.Human polyclonal Tregs were obtained by sorting CD4+CD25+CD127lo cellsby FACS from total PBMCs, followed by in vitro stimulation withanti-CD3/CD28 coated beads in the presence of IL-2 during 12-13 days, asdescribed (E. Gauthy et al. PLoS One 8, e76186 (2013).). 293T cells weretransiently transfected with hGARP- and hTGF-β1-encoding plasmids usingthe TransIT-LT1 transfection Reagent (Mirus Bio).

Generation of MHG mAbs

DBA/2 or Balb/c mice were immunized with live P1.HTR+hGARP cells,following a previously described injection scheme (M. M. Lemaire, et al.J. Immunol. Methods, (2011)). Lymphocytes from mice with high titers ofanti-hGARP antibodies, as determined by FACS, were fused to SP2/neocells in the presence of polyethylene glycol. Hybridomas were selectedin HAT medium and cloned under limiting dilution conditions.Supernatants of hybridoma clones were screened by FACS for the presenceof antibodies binding to BW5147+hGARP cells. Fourteen positive cloneswere selected, further subcloned to ensure clonality, and amplified forlarge scale-production and purification of 14 new anti-hGARP mAbs (MHG-1to -14).

Generation of LHG mAbs

Immunizations of llamas, harvesting of peripheral blood lymphocytes(PBLs), RNA preparation and amplification of antibody fragments wereperformed as described (C. Basilico, et al. J. Clin. Invest. 124,3172-3186 (2014)). Briefly, four llamas were injected six times atweekly intervals with 10⁷ BW5147+hGARP+hTGFB1 cells and Freund'sincomplete adjuvant in two regions of the neck muscles located a fewcentimeters apart. Another four llamas were injected four times biweeklywith a mix of plasmids containing hGARP cDNA and hTGFB1 cDNA,respectively. Blood samples (10 ml) were collected to monitor IgG1responses against hGARP/TGF-β1 complexes by ELISA, using immobilizedrecombinant GARP/TGF-β1 complexes (produced in HEK-293E cellsco-transfected with hTGFB1 and hGARP truncated from thetransmembrane-coding region) for capture, followed by a mouse anti-llamaIgG1 antibody (clone 27E10) and a HRP-conjugated donkey anti-mouseantibody (Jackson) for detection. Three-to-four days after the lastimmunization, 400 ml of blood were collected from responding llamas,PBLs were isolated on a Ficoll-Paque gradient and total RNA wasextracted as described (P. Chomczynski, et al. Anal. Biochem. 162,156-159 (1987)). On average, 450 μg of RNA were obtained and used forrandom cDNA synthesis followed by PCR amplification of theimmunoglobulin heavy and light chain variable regions (VH, Vλ and Vκ).Two independent phagemid libraries, coding for VH/Vλ and VH/Vκ Fabs,respectively, were constructed as previously described (C. Basilico, etal. J. Clin. Invest. 124, 3172-3186 (2014).) to obtain a diversity of1-7×10⁸ Fabs in each library. Phages expressing Fabs were produced andselected according to standard protocols. Briefly, 2 to 3 rounds ofphage selections were performed by binding on immobilized recombinantGARP/TGF-β1, washing and elution with trypsin. In some instances,counter selections with soluble hGARP (hGARP1-628 fused to aTEV-3×StrepTag produced in 293E cells) and soluble latent TGF-β1 wereused to enrich for Fabs binding hGARP/TGF-β1 complexes only. Individualcolonies were isolated and periplasmic fractions containing soluble Fabswere produced by IPTG induction. Fabs in periplasmic fractions were thenscreened by ELISA for binding to immobilized hGARP/TGF-β1. VH and VLregions of Fab clones binding to hGARP/TGF-β1 complexes were sequenced.Fab clones were divided into 17 families, based on similarities in thesequences coding for the VH CDR3 region. VH and VL sequences from onerepresentative clone of each family were subcloned in a full human IgG1backbone, and the resulting plasmids were transfected into HEK-293Ecells to produce and purify 17 new anti-hGARP mAbs (LHG-1 to -17).

Analysis of FOXP3i1 Methylation

Proportions of cells with a demethylated FOXP3i1 in human PBMCs, inhuman polyclonal Treg populations or in splenocytes from NSG micegrafted with human cells were measured by methyl-specific qPCR asdescribed (I. J. de Vries, et al. Clin. Cancer Res. 17, 841-848 (2011)),using the following primers (sense, antisense and Taqman probe, 5′-3′,with underlined nucleotides corresponding to LNA® modified bases): totalFOXP3i1 alleles: AAACCTACTACAAAACAAAACAAC (SEQ ID NO:56)/GGAGGAAGAGAAGAGGGTA (SEQ ID NO: 57)/CCTATAAAATAAAATATCTACCCTC (SEQID NO: 58); demethylated FOXP3i1 alleles: TCTACCCTCTTCTCTTCCTCCA (SEQ IDNO: 59)/GATTTTTTTGTTATTGATGTTATGGT (SEQ ID NO: 60)/AAACCCAACACATCCAACCA(SEQ ID NO: 61).

Assay to Measure Active TGF-β1 Production by Human Treg Cells

A human Treg clone (10⁶ cells/ml) was stimulated in serum-free mediumwith coated anti-CD3 (Orthoclone OKT3; Janssen-Cilag, 1 μg/ml) andsoluble anti-CD28 (BD Biosciences; 1 μg/ml), in the presence or absenceof 10 μg/ml of an anti-hGARP mAb (clones tested: MHG-1 to -14; LHG-1 to-17; Plato-1 from Enzo Life Sciences; 272G6 and 50G10 from SynapticSystems; 7B11 from BioLegend) or of an anti-hTGF-β13 antibody (clone1D11, R&D systems). Cells were lysed after 24 hours and submitted toSDS-PAGE under reducing conditions. Gels were blotted on nitrocellulosemembranes with the iBlot system (Life Technologies). After blocking,membranes were incubated with primary antibodies directed againstphosphorylated SMAD2 (pSMAD2, Cell Signaling Technologies) or β-ACTIN(SIGMA), then with secondary HRP coupled antibodies and revealed with anECL substrate (ThermoFisher Scientific). The presence of pSMAD2indicates production of active TGF-β1 by the stimulated Treg clone. ECLsignals were quantified by measuring the density of the 55 kDa pSMAD2and 40 kDa 13-ACTIN bands on autoradiographs, using the Image Jsoftware.

Flow Cytometry

Intact or permeabilized cells were labeled according to standardprotocols, using combinations of the following primary and/or secondaryreagents as indicated in the figures. Primary antibodies: biotinylatedMHG-1 to 14; LHG-1 to -17; anti-hGARP clone Plato 1 (Enzo LifeSciences); antihCD45-PerCP, anti-hCD3-FITC or anti-hCD3-APC,anti-hCD4-FITC or anti-hCD4-APC, antihCD45RA-PE-Cy7 (Biolegend);anti-hCD8α-APC-H7, anti-CD25-PE-Cy7, anti-hCD127-PE (BD Biosciences);anti-hFOXP3-PE or anti-hFOXP3-APC (eBiosciences); anti-hLAP-APC (R&DSystems); anti-HA (Eurogentec). Secondary antibodies or reagents:anti-hIgG1-biotine (Jackson ImmunoResearch); anti-mIgG1-AF647,anti-mIgG2b-AF647, LIVE/DEAD Fixable Near-IR Dead cell stain kit (LifeTechnologies); Streptavidine-PE (BD Biosciences). Labeled cells wereanalyzed on a LSR Fortessa cytometer or sorted on a FACSARIA III (bothfrom BD Biosciences), and results were computed with the FlowJo Software(Treestar).

In Vitro Suppression Assays

2×10⁴ Th cells were seeded alone or with the indicated numbers of Tregs,and stimulated with coated anti-CD3 (Orthoclone OKT3, Janssen-Cilag, 1μg/ml) and soluble anti-CD28 (BDBiosciences, 1 μg/ml), in the presenceor absence of 10 μg/ml of an anti-hGARP mAb (MHG or LHG), an anti-TGF-bantibody (clone 1D11, R&D Systems) or an isotype control (mIgG1 clone11711, R&D Systems). [methyl-3H]Thymidine (0.5 mCi/well) was addedduring the last 16 hours of a 4 day-culture.

Xenogeneic Graft-Versus-Host Disease in NSG Mice

NSG mice were irradiated (1.5 Gy) one day before tail vein injections ofhuman PBMCs (3×10⁶ per mouse) alone, or mixed with autologous polyclonalTregs (1.5×10⁶ per mouse). One day before graft and weekly thereafter,mice received i.p. injections of PBS or 400 μg of MHG-8 (mIgG1), ananti-TGF-b1 antibody (mIgG1 clone 13A1/A26) or an isotype control (mIgG1anti-TNP clone B8401H5.M). Mice were monitored bi-weekly for thedevelopment of GVHD. A global disease score was established by adding upscores attributed in the presence of the following symptoms: weight loss(1 if >10%; 2 if >20%); anemia or icterus (1 if white or yellow ears; 2if white or yellow ears and tail); humped posture (1); reduced activity(1 if limited activity; 2 if no activity); hair loss (1). Mice wereeuthanized when reaching a global score >6. Death corresponds to amaximum score of 8.

Cytokine Concentrations in Sera

Concentrations of human IL-2, IL-10, and IFNg in mouse serum weredetermined using a Bio-Plex Pro Human Cytokine 17-plex Assay accordingto the manufacturer's recommendations (Bio-Rad Laboratories). Limits ofdetection in this assay were: 0.12 pg/ml for IL-2; 1.56 μg/ml for IFNγ;2.48 μg/ml for IL-10.

EQUIVALENTS

The disclosure may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the disclosure. Scope of the disclosure is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are therefore intended to be embraced herein.

1-24. (canceled)
 25. A method of producing an anti-GARP/TGF-β1 antibodycomprising: a) culturing a host cell comprising one or more expressionvectors encoding an anti-GARP/TGF-β1 antibody selected by: (1) obtainingat least one antibody that binds to a human GARP/TGF-β1 complex; (2)determining whether the at least one antibody of (1) has the propertiesof: (z) inhibiting release of active TGF-β1 from the GARP/TGF-β complex;(y) an absence of binding to GARP that is not complexed with TGF-β1; (x)an absence of binding to active TGF-β1; and (w) binding to a mixedconformational epitope comprising amino acids from both GARP and TGF-β1,wherein the mixed conformational epitope comprises at least one aminoacid from the Latency Associated Peptide (LAP) of TGF-β1 selected fromthe group of residues 58, 100, 146, 269, 270, 271, 272, and 273 ofTGF-β1 (SEQ ID NO:53); and at least one residue from mature TGF-β1selected from the group of residues 284, 336, 337, 338, 341, and 345 ofTGF-β1 (SEQ ID NO:53), and (3) selecting the anti-GARP/TGF-β1 antibodyhaving the properties: (z), (y), (x), and (w), and b) isolating theselected anti-GARP/TGF-β1 antibody.
 26. The method of claim 25, whereinthe mixed conformational epitope comprises residues 137, 138 and 139 ofGARP (SEQ ID NO: 1); and at least one residue selected from the group ofresidues 113, 114, 116, 117, 118, 119, 140, 142, 143, 144, 145, 146,162, 163, 165, 166, 167, 170 and 189 of GARP (SEQ ID NO: 1).
 27. Themethod of claim 25, wherein the mixed conformational epitope comprisesresidues 137, 138 and 139 of GARP (SEQ ID NO: 1); at least one residueselected from the group of residues 162 and 163 of GARP (SEQ ID NO: 1);residue 58 from the Latency Associated Peptide (LAP) of TGF-β1 (SEQ IDNO:53); and residue 338 from mature TGF-β1 (SEQ ID NO:53).
 28. Themethod of claim 25, wherein the anti-GARP/TGF-β1 antibody is a humanizedantibody.
 29. A method of producing an anti-GARP/TGF-β1 antibodycomprising: a) culturing a host cell comprising one or more expressionvectors encoding CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3 ofa donor anti-GARP/TGF-β1 antibody selected by: (1) obtaining at leastone antibody that binds to a human GARP/TGF-β1 complex; (2) determiningwhether the at least one antibody of (1) has the properties of: (z)inhibiting release of active TGF-β1 from the GARP/TGF-β complex; (y) anabsence of binding to GARP that is not complexed with TGF-β1; (x) anabsence of binding to active TGF-β1; and (w) binding to a mixedconformational epitope comprising amino acids from both GARP and TGF-β1,wherein the mixed conformational epitope comprises at least one aminoacid from the Latency Associated Peptide (LAP) of TGF-β1 selected fromthe group of residues 58, 100, 146, 269, 270, 271, 272, and 273 ofTGF-β1 (SEQ ID NO:53); and at least one residue from mature TGF-β1selected from the group of residues 284, 336, 337, 338, 341, and 345 ofTGF-β1 (SEQ ID NO:53), and (3) selecting the donor anti-GARP/TGF-β1antibody having the properties: (z), (y), (x), and (w), and b) isolatingthe anti-GARP/TGF-β1 antibody having CDRH1, CDRH2, CDRH3, CDRL1, CDRL2,and CDRL3 of the donor antibody.
 30. The method of claim 29, wherein themixed conformational epitope comprises residues 137, 138 and 139 of GARP(SEQ ID NO: 1); and at least one residue selected from the group ofresidues 113, 114, 116, 117, 118, 119, 140, 142, 143, 144, 145, 146,162, 163, 165, 166, 167, 170 and 189 of GARP (SEQ ID NO: 1).
 31. Themethod of claim 29, wherein the mixed conformational epitope comprisesresidues 137, 138 and 139 of GARP (SEQ ID NO: 1); and at least oneresidue selected from the group of residues 162 and 163 of GARP (SEQ IDNO: 1).
 32. The method of claim 29, wherein the anti GARP/TGF-β1antibody is a humanized antibody.